John Boothroyd

All Publications

• Emergent actin flows explain distinct modes of gliding motility NATURE PHYSICS Hueschen, C. L., Segev-Zarko, L., Chen, J., Legros, M. A., Larabell, C. A., Boothroyd, J. C., Phillips, R., Dunn, A. R. 2024

  View details for DOI 10.1038/s41567-024-02652-4

  View details for Web of Science ID 001331252700003

• Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum. mBio Sun, S. Y., Segev-Zarko, L., Pintilie, G. D., Kim, C. Y., Staggers, S. R., Schmid, M. F., Egan, E. S., Chiu, W., Boothroyd, J. C. 2024: e0286423

  Abstract

  Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells.Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

  View details for DOI 10.1128/mbio.02864-23

  View details for PubMedID 38456679

• Cryo-electron tomography with mixed-scale dense neural networks reveals key steps in deployment of Toxoplasma invasion machinery. PNAS nexus Segev-Zarko, L. A., Dahlberg, P. D., Sun, S. Y., Pelt, D. M., Kim, C. Y., Egan, E. S., Sethian, J. A., Chiu, W., Boothroyd, J. C. 2022; 1 (4): pgac183

  Abstract

  Host cell invasion by intracellular, eukaryotic parasites within the phylum Apicomplexa is a remarkable and active process involving the coordinated action of apical organelles and other structures. To date, capturing how these structures interact during invasion has been difficult to observe in detail. Here, we used cryogenic electron tomography to image the apical complex of Toxoplasma gondii tachyzoites under conditions that mimic resting parasites and those primed to invade through stimulation with calcium ionophore. Through the application of mixed-scale dense networks for image processing, we developed a highly efficient pipeline for annotation of tomograms, enabling us to identify and extract densities of relevant subcellular organelles and accurately analyze features in 3-D. The results reveal a dramatic change in the shape of the anteriorly located apical vesicle upon its apparent fusion with a rhoptry that occurs only in the stimulated parasites. We also present information indicating that this vesicle originates from the vesicles that parallel the intraconoidal microtubules and that the latter two structures are linked by a novel tether. We show that a rosette structure previously proposed to be involved in rhoptry secretion is associated with apical vesicles beyond just the most anterior one. This result, suggesting multiple vesicles are primed to enable rhoptry secretion, may shed light on the mechanisms Toxoplasma employs to enable repeated invasion attempts. Using the same approach, we examine Plasmodium falciparum merozoites and show that they too possess an apical vesicle just beneath a rosette, demonstrating evolutionary conservation of this overall subcellular organization.

  View details for DOI 10.1093/pnasnexus/pgac183

  View details for PubMedID 36329726

  View details for PubMedCentralID PMC9615128

• Cryo-ET of Toxoplasma parasites gives subnanometer insight into tubulin-based structures. Proceedings of the National Academy of Sciences of the United States of America Sun, S. Y., Segev-Zarko, L., Chen, M., Pintilie, G. D., Schmid, M. F., Ludtke, S. J., Boothroyd, J. C., Chiu, W. 2022; 119 (6)

  Abstract

  Tubulin is a conserved protein that polymerizes into different forms of filamentous structures in Toxoplasma gondii, an obligate intracellular parasite in the phylum Apicomplexa. Two key tubulin-containing cytoskeletal components are subpellicular microtubules (SPMTs) and conoid fibrils (CFs). The SPMTs help maintain shape and gliding motility, while the CFs are implicated in invasion. Here, we use cryogenic electron tomography to determine the molecular structures of the SPMTs and CFs in vitrified intact and detergent-extracted parasites. Subvolume densities from detergent-extracted parasites yielded averaged density maps at subnanometer resolutions, and these were related back to their architecture in situ. An intralumenal spiral lines the interior of the 13-protofilament SPMTs, revealing a preferred orientation of these microtubules relative to the parasite's long axis. Each CF is composed of nine tubulin protofilaments that display a comma-shaped cross-section, plus additional associated components. Conoid protrusion, a crucial step in invasion, is associated with an altered pitch of each CF. The use of basic building blocks of protofilaments and different accessory proteins in one organism illustrates the versatility of tubulin to form two distinct types of assemblies, SPMTs and CFs.

  View details for DOI 10.1073/pnas.2111661119

  View details for PubMedID 35121661

• Proximity-Labeling Reveals Novel Host and Parasite Proteins at the Toxoplasma Parasitophorous Vacuole Membrane. mBio Cygan, A. M., Jean Beltran, P. M., Mendoza, A. G., Branon, T. C., Ting, A. Y., Carr, S. A., Boothroyd, J. C. 2021: e0026021

  Abstract

  Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCE Toxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.

  View details for DOI 10.1128/mBio.00260-21

  View details for PubMedID 34749525

• Coimmunoprecipitation with MYR1 Identifies Three Additional Proteins within the Toxoplasma gondii Parasitophorous Vacuole Required for Translocation of Dense Granule Effectors into Host Cells. mSphere Cygan, A. M., Theisen, T. C., Mendoza, A. G., Marino, N. D., Panas, M. W., Boothroyd, J. C. 2020; 5 (1)

  Abstract

  Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV), in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54), and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16 and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5 processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins.IMPORTANCE Toxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells and provide additional potential drug targets.

  View details for DOI 10.1128/mSphere.00858-19

  View details for PubMedID 32075880

• A single-parasite transcriptional atlas of Toxoplasma gondii reveals novel control of antigen expression. eLife Xue, Y., Theisen, T. C., Rastogi, S., Ferrel, A., Quake, S. R., Boothroyd, J. C. 2020; 9

  Abstract

  Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.

  View details for DOI 10.7554/eLife.54129

  View details for PubMedID 32065584

• Identification of a novel protein complex essential for effector translocation across the parasitophorous vacuole membrane of Toxoplasma gondii PLOS PATHOGENS Marino, N. D., Panas, M. W., Franco, M., Theisen, T. C., Naor, A., Rastogi, S., Buchholz, K. R., Lorenzi, H. A., Boothroyd, J. C. 2018; 14 (1): e1006828

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite that can infect virtually all nucleated cells in warm-blooded animals. The ability of Toxoplasma tachyzoites to infect and successfully manipulate its host is dependent on its ability to transport "GRA" proteins that originate in unique secretory organelles called dense granules into the host cell in which they reside. GRAs have diverse roles in Toxoplasma's intracellular lifecycle, including co-opting crucial host cell functions and proteins, such as the cell cycle, c-Myc and p38 MAP kinase. Some of these GRA proteins, such as GRA16 and GRA24, are secreted into the parasitophorous vacuole (PV) within which Toxoplasma replicates and are transported across the PV membrane (PVM) into the host cell, but the translocation process and its machinery are not well understood. We previously showed that TgMYR1, which is cleaved by TgASP5 into two fragments, localizes to the PVM and is essential for GRA transport into the host cell. To identify additional proteins necessary for effector transport, we screened Toxoplasma mutants defective in c-Myc up-regulation for their ability to export GRA16 and GRA24 to the host cell nucleus. Here we report that novel proteins MYR2 and MYR3 play a crucial role in translocation of a subset of GRAs into the host cell. MYR2 and MYR3 are secreted into the PV space and co-localize with PV membranes and MYR1. Consistent with their predicted transmembrane domains, all three proteins are membrane-associated, and MYR3, but not MYR2, stably associates with MYR1, whose N- and C-terminal fragments are disulfide-linked. We further show that fusing intrinsically disordered effectors to a structured DHFR domain blocks the transport of other effectors, consistent with a translocon-based model of effector transport. Overall, these results reveal a novel complex at the PVM that is essential for effector translocation into the host cell.

  View details for PubMedID 29357375

  View details for PubMedCentralID PMC5794187

• Toxoplasma protein export and effector function. Nature microbiology Seizova, S., Ferrel, A., Boothroyd, J., Tonkin, C. J. 2024

  Abstract

  Toxoplasma gondii is a single-celled eukaryotic parasite with a considerable host range that must invade the cells of warm-blooded hosts to survive and replicate. The challenges and opportunities that such a strategy represent have been met by the evolution of effectors that are delivered into host cells, counter host defences and co-opt host cell functions for their own purposes. These effectors are delivered in two waves using distinct machinery for each. In this Review, we focus on understanding the architecture of these protein-export systems and how their protein cargo is recognized and selected. We discuss the recent findings on the role that host manipulation has in latent Toxoplasma infections. We also discuss how these recent findings compare to protein export in the related Plasmodium spp. (the causative agent of malaria) and how this can inform our understanding of host manipulation in the larger Apicomplexa phylum and its evolution.

  View details for DOI 10.1038/s41564-023-01563-z

  View details for PubMedID 38172621

  View details for PubMedCentralID 3109627

• Host MOSPD2 enrichment at the parasitophorous vacuole membrane varies between Toxoplasma strains and involves complex interactions. mSphere Ferrel, A., Romano, J., Panas, M. W., Coppens, I., Boothroyd, J. C. 2023: e0067022

  Abstract

  Toxoplasma gondii is an obligate, intracellular parasite. Infection of a cell produces a unique niche for the parasite named the parasitophorous vacuole (PV) initially composed of host plasma membrane invaginated during invasion. The PV and its membrane (parasitophorous vacuole membrane [PVM]) are subsequently decorated with a variety of parasite proteins allowing the parasite to optimally grow in addition to manipulate host processes. Recently, we reported a proximity-labeling screen at the PVM-host interface and identified host endoplasmic reticulum (ER)-resident motile sperm domain-containing protein 2 (MOSPD2) as being enriched at this location. Here we extend these findings in several important respects. First, we show that the extent and pattern of host MOSPD2 association with the PVM differ dramatically in cells infected with different strains of Toxoplasma. Second, in cells infected with Type I RH strain, the MOSPD2 staining is mutually exclusive with regions of the PVM that associate with mitochondria. Third, immunoprecipitation and liquid chromatography tandem mass spectrometry (LC-MS/MS) with epitope-tagged MOSPD2-expressing host cells reveal strong enrichment of several PVM-localized parasite proteins, although none appear to play an essential role in MOSPD2 association. Fourth, most MOSPD2 associating with the PVM is newly translated after infection of the cell and requires the major functional domains of MOSPD2, identified as the CRAL/TRIO domain and tail anchor, although these domains were not sufficient for PVM association. Lastly, ablation of MOSPD2 results in, at most, a modest impact on Toxoplasma growth in vitro. Collectively, these studies provide new insight into the molecular interactions involving MOSPD2 at the dynamic interface between the PVM and the host cytosol. IMPORTANCE Toxoplasma gondii is an intracellular pathogen that lives within a membranous vacuole inside of its host cell. This vacuole is decorated by a variety of parasite proteins that allow it to defend against host attack, acquire nutrients, and interact with the host cell. Recent work identified and validated host proteins enriched at this host-pathogen interface. Here, we follow up on one candidate named MOSPD2 shown to be enriched at the vacuolar membrane and describe it as having a dynamic interaction at this location depending on a variety of factors. Some of these include the presence of host mitochondria, intrinsic domains of the host protein, and whether translation is active. Importantly, we show that MOSPD2 enrichment at the vacuole membrane differs between strains indicating active involvement of the parasite with this phenotype. Altogether, these results shed light on the mechanism and role of protein associations in the host-pathogen interaction.

  View details for DOI 10.1128/msphere.00670-22

  View details for PubMedID 37341482

• Actin self-organization in gliding parasitic cells Hueschen, C. L., Zarko, L., Chen, J., LeGros, M., Larabell, C. A., Boothroyd, J. C., Phillips, R., Dunn, A. R. CELL PRESS. 2023: 5A

  View details for Web of Science ID 000989629700026

• Emergent actin flows explain distinct modes of gliding motility Hueschen, C. L., Zarko, L., Chen, J., LeGros, M. A., Larabell, C. A., Boothroyd, J. C., Phillips, R., Dunn, A. R. AMER SOC CELL BIOLOGY. 2023: 1160

  View details for Web of Science ID 001051001304292

• Transcriptional signatures of clonally derived Toxoplasma tachyzoites reveal novel insights into the expression of a family of surface proteins. PloS one Theisen, T. C., Boothroyd, J. C. 2022; 17 (2): e0262374

  Abstract

  Toxoplasma gondii has numerous, large, paralogous gene families that are likely critical for supporting its unparalleled host range: nearly any nucleated cell in almost any warm-blooded animal. The SRS (SAG1-related sequence) gene family encodes over 100 proteins, the most abundant of which are thought to be involved in parasite attachment and, based on their stage-specific expression, evading the host immune response. For most SRS proteins, however, little is understood about their function and expression profile. Single-parasite RNA-sequencing previously demonstrated that across an entire population of lab-grown tachyzoites, transcripts for over 70 SRS genes were detected in at least one parasite. In any one parasite, however, transcripts for an average of only 7 SRS genes were detected, two of which, SAG1 and SAG2A, were extremely abundant and detected in virtually all. These data do not address whether this pattern of sporadic SRS gene expression is consistently inherited among the progeny of a given parasite or arises independently of lineage. We hypothesized that if SRS expression signatures are stably inherited by progeny, subclones isolated from a cloned parent would be more alike in their expression signatures than they are to the offspring of another clone. In this report, we compare transcriptomes of clonally derived parasites to determine the degree to which expression of the SRS family is stably inherited in individual parasites. Our data indicate that in RH tachyzoites, SRS genes are variably expressed even between parasite samples subcloned from the same parent within approximately 10 parasite divisions (72 hours). This suggests that the pattern of sporadically expressed SRS genes is highly variable and not driven by inheritance mechanisms, at least under our conditions.

  View details for DOI 10.1371/journal.pone.0262374

  View details for PubMedID 35213559

• Seizing control: how dense granule effector proteins enable Toxoplasma to take charge. Molecular microbiology Panas, M. W., Boothroyd, J. C. 2021

  Abstract

  Control of the host cell is crucial to the Apicomplexan parasite, Toxoplasma gondii, while it grows intracellularly. To achieve this goal, these single-celled eukaryotes export a series of effector proteins from organelles known as "dense granules" that interfere with normal cellular processes and responses to invasion. While some effectors are found attached to the outer surface of the parasitophorous vacuole (PV) in which Toxoplasma tachyzoites reside, others are found in the host cell's cytoplasm and yet others make their way into the host nucleus, where they alter host transcription. Among the processes that are severely altered are innate immune responses, host cell cycle and association with host organelles. The ways in which these crucial processes are altered through the coordinated action of a large collection of effectors is as elegant as it is complex, and is the central focus of the following review; we also discuss recent advances in our understanding of how dense granule effector proteins are trafficked out of the PV.

  View details for DOI 10.1111/mmi.14679

  View details for PubMedID 33400323

• Toxoplasma Uses GRA16 To Upregulate Host c-Myc. mSphere Panas, M. W., Boothroyd, J. C. 2020; 5 (3)

  Abstract

  Manipulation of the host cell is a crucial part of life for many intracellular organisms. We have recently come to appreciate the extent to which the intracellular pathogen Toxoplasma gondii reprograms its host cell, and this is illustrated by the marked upregulation of the central regulator c-Myc, an oncogene that coordinates myriad cellular functions. In an effort to identify an effector protein capable of regulating c-Myc, our laboratory constructed a screen for mutant parasites unable to accomplish this upregulation. Interestingly, this screen identified numerous components of a complex located in/on the parasitophorous vacuole membrane necessary to translocate Toxoplasma proteins out into the host cytosol, but it never identified a specific effector protein. Thus, how the parasite upregulates c-Myc has largely been a mystery. Previously, the Toxoplasma dense granule protein GRA16 has been described to bind to one isoform of PP2A-B, a regulatory subunit that coordinates the activity of the catalytic protein phosphatase PP2A. As other PP2A subunits have been reported to target PP2A protein phosphatase activity to c-Myc, subsequently leading to c-Myc destabilization, we examined whether GRA16 has an impact on host c-Myc accumulation. Expression of Toxoplasma's GRA16 protein in Neospora caninum, a close relative of Toxoplasma that does not naturally upregulate host c-Myc, conferred the ability on Neospora to do this now. Further support was obtained by deleting the GRA16 gene from Toxoplasma and observing a severely diminished ability of Toxoplasma tachyzoites to upregulate host c-Myc. Thus, GRA16 is an effector protein central to Toxoplasma's ability to upregulate host c-Myc.IMPORTANCE The proto-oncogene c-Myc plays a crucial role in the growth and division of many animal cells. Previous studies have identified an active upregulation of c-Myc by Toxoplasma tachyzoites, suggesting the existence of one or more exported "effector" proteins. The identity of such an effector, however, has not previously been known. Here, we show that a previously known secreted protein, GRA16, plays a crucial role in c-Myc upregulation. This finding will enable further dissection of the precise mechanism and role of c-Myc upregulation in Toxoplasma-infected cells.

  View details for DOI 10.1128/mSphere.00402-20

  View details for PubMedID 32581080

• Differential Impacts on Host Transcription by ROP and GRA Effectors from the Intracellular Parasite Toxoplasma gondii. mBio Rastogi, S., Xue, Y., Quake, S. R., Boothroyd, J. C. 2020; 11 (3)

  Abstract

  The intracellular parasite Toxoplasma gondii employs a vast array of effector proteins from the rhoptry and dense granule organelles to modulate host cell biology; these effectors are known as ROPs and GRAs, respectively. To examine the individual impacts of ROPs and GRAs on host gene expression, we developed a robust, novel protocol to enrich for ultrapure populations of a naturally occurring and reproducible population of host cells called uninfected-injected (U-I) cells, which Toxoplasma injects with ROPs but subsequently fails to invade. We then performed single-cell transcriptomic analysis at 1 to 3h postinfection on U-I cells (as well as on uninfected and infected controls) arising from infection with either wild-type parasites or parasites lacking the MYR1 protein, which is required for soluble GRAs to cross the parasitophorous vacuole membrane (PVM) and reach the host cell cytosol. Based on comparisons of infected and U-I cells, the host's earliest response to infection appears to be driven primarily by the injected ROPs, which appear to induce immune and cellular stress pathways. These ROP-dependent proinflammatory signatures appear to be counteracted by at least some of the MYR1-dependent GRAs and may be enhanced by the MYR-independent GRAs (which are found embedded within the PVM). Finally, signatures detected in uninfected bystander cells from the infected monolayers suggest that MYR1-dependent paracrine effects also counteract inflammatory ROP-dependent processes.IMPORTANCE This work performs transcriptomic analysis of U-I cells, captures the earliest stage of a host cell's interaction with Toxoplasma gondii, and dissects the effects of individual classes of parasite effectors on host cell biology.

  View details for DOI 10.1128/mBio.00182-20

  View details for PubMedID 32518180

• What a Difference 30Years Makes! A Perspective on Changes in Research Methodologies Used to Study Toxoplasma gondii. Methods in molecular biology (Clifton, N.J.) Boothroyd, J. C. 2020; 2071: 1–25

  Abstract

  Toxoplasma gondii is a remarkable species with a rich cell, developmental, and population biology. It is also sometimes responsible for serious disease in animals and humans and the stages responsible for such disease are relatively easy to study in vitro or in laboratory animal models. As a result of all this, Toxoplasma has become the subject of intense investigation over the last several decades, becoming a model organism for the study of the phylum of which it is a member, Apicomplexa. This has led to an ever-growing number of investigators applying an ever-expanding set of techniques to dissecting how Toxoplasma "ticks" and how it interacts with its many hosts. In this perspective piece I first wind back the clock 30years and then trace the extraordinary pace of methodologies that have propelled the field forward to where we are today. In keeping with the theme of this collection, I focus almost exclusively on the parasite, rather than host side of the equation. I finish with a few thoughts about where the field might be headed-though if we have learned anything, the only sure prediction is that the pace of technological advance will surely continue to accelerate and the future will give us still undreamed of methods for taking apart (and then putting back together) this amazing organism with all its intricate biology. We have so far surely just scratched the surface.

  View details for DOI 10.1007/978-1-4939-9857-9_1

  View details for PubMedID 31758444

• Effectors produced by rhoptries and dense granules: an intense conversation between parasite and host in many languages TOXOPLASMA GONDII: THE MODEL APICOMPLEXAN-PERSPECTIVES AND METHODS, 3RD EDITION Boothroyd, J. C., Hakimi, M., Weiss, L. M., Kim, K. 2020: 789–806

  View details for DOI 10.1016/B978-0-12-815041-2.00017-7

  View details for Web of Science ID 000558957200018

• Translocation of effector proteins into host cells by Toxoplasma gondii. Current opinion in microbiology Rastogi, S., Cygan, A. M., Boothroyd, J. C. 2019; 52: 130–38

  Abstract

  The Apicomplexan parasite, Toxoplasma gondii, is an obligate intracellular organism that must co-opt its host cell to survive. To this end, Toxoplasma parasites introduce a suite of effector proteins from two secretory compartments called rhoptries and dense granules into the host cells. Once inside, these effectors extensively modify the host cell to facilitate parasite penetration, replication and persistence. In this review, we summarize the most recent advances in current understanding of effector translocation from Toxoplasma's rhoptry and dense granule organelles into the host cell, with comparisons to Plasmodium spp. for broader context.

  View details for DOI 10.1016/j.mib.2019.07.002

  View details for PubMedID 31446366

• Translocation of Dense Granule Effectors across the Parasitophorous Vacuole Membrane in Toxoplasma-Infected Cells Requires the Activity of ROP17, a Rhoptry Protein Kinase. mSphere Panas, M. W., Ferrel, A., Naor, A., Tenborg, E., Lorenzi, H. A., Boothroyd, J. C. 2019; 4 (4)

  Abstract

  Toxoplasma gondii tachyzoites co-opt host cell functions through introduction of a large set of rhoptry- and dense granule-derived effector proteins. These effectors reach the host cytosol through different means: direct injection for rhoptry effectors and translocation across the parasitophorous vacuolar membrane (PVM) for dense granule (GRA) effectors. The machinery that translocates these GRA effectors has recently been partially elucidated, revealing three components, MYR1, MYR2, and MYR3. To determine whether other proteins might be involved, we returned to a library of mutants defective in GRA translocation and selected one with a partial defect, suggesting it might be in a gene encoding a new component of the machinery. Surprisingly, whole-genome sequencing revealed a missense mutation in a gene encoding a known rhoptry protein, a serine/threonine protein kinase known as ROP17. ROP17 resides on the host cytosol side of the PVM in infected cells and has previously been known for its activity in phosphorylating and thereby inactivating host immunity-related GTPases. Here, we show that null or catalytically dead mutants of ROP17 are defective in GRA translocation across the PVM but that translocation can be rescued "in trans" by ROP17 delivered by other tachyzoites infecting the same host cell. This strongly argues that ROP17's role in regulating GRA translocation is carried out on the host cytosolic side of the PVM, not within the parasites or lumen of the parasitophorous vacuole. This represents an entirely new way in which the different secretory compartments of Toxoplasma tachyzoites collaborate to modulate the host-parasite interaction.IMPORTANCE When Toxoplasma infects a cell, it establishes a protective parasitophorous vacuole surrounding it. While this vacuole provides protection, it also serves as a barrier to the export of parasite effector proteins that impact and take control of the host cell. Our discovery here that the parasite rhoptry protein ROP17 is necessary for export of these effector proteins provides a distinct, novel function for ROP17 apart from its known role in protecting the vacuole. This will enable future research into ways in which we can prevent the export of effector proteins, thereby preventing Toxoplasma from productively infecting its animal and human hosts.

  View details for DOI 10.1128/mSphere.00276-19

  View details for PubMedID 31366709

• Toxoplasma Controls Host Cyclin E Expression through the Use of a Novel MYR1-Dependent Effector Protein, HCE1. mBio Panas, M. W., Naor, A., Cygan, A. M., Boothroyd, J. C. 2019; 10 (2)

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite that establishes a favorable environment in the host cells in which it replicates. We have previously reported that it uses MYR-dependent translocation of dense granule proteins to elicit a key set of host responses related to the cell cycle, specifically, E2F transcription factor targets, including cyclin E. We report here the identification of a novel Toxoplasma effector protein that is exported from the parasitophorous vacuole in a MYR1-dependent manner and localizes to the host's nucleus. Parasites lacking this inducer of host cyclin E (HCE1) are unable to modulate E2F transcription factor target genes and exhibit a substantial growth defect. Immunoprecipitation of HCE1 from infected host cells showed that HCE1 efficiently binds elements of the cyclin E regulatory complex, namely, DP1 and its partners E2F3 and E2F4. Expression of HCE1 in Neospora caninum, or in uninfected human foreskin fibroblasts (HFFs), showed localization of the expressed protein to the host nuclei and strong cyclin E upregulation. Thus, HCE1 is a novel effector protein that is necessary and sufficient to impact the E2F axis of transcription, resulting in co-opting of host functions to the advantage of Toxoplasma IMPORTANCE Like most Apicomplexan parasites, Toxoplasma gondii has the remarkable ability to invade and establish a replicative niche within another eukaryotic cell, in this case, any of a large number of cell types in almost any warm-blooded animals. Part of the process of establishing this niche is the export of effector proteins to co-opt host cell functions in favor of the parasite. Here we identify a novel effector protein, HCE1, that the parasites export into the nucleus of human cells, where it modulates the expression of multiple genes, including the gene encoding cyclin E, one of the most crucial proteins involved in controlling when and whether a human cell divides. We show that HCE1 works through binding to specific transcription factors, namely, E2F3, E2F4, and DP1, that normally carefully regulate these all-important pathways. This represents a new way in which these consummately efficient infectious agents co-opt the human cells that they so efficiently grow within.

  View details for DOI 10.1128/mBio.00674-19

  View details for PubMedID 31040242

• Erratum for Franco et al., "A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells". mBio Franco, M., Panas, M. W., Marino, N. D., Lee, M. W., Buchholz, K. R., Kelly, F. D., Bednarski, J. J., Sleckman, B. P., Pourmand, N., Boothroyd, J. C. 2018; 9 (5)

  View details for PubMedID 30279290

• A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions MOLECULAR MICROBIOLOGY Blank, M. L., Parker, M. L., Ramaswamy, R., Powell, C. J., English, E. D., Adomako-Ankomah, Y., Pernas, L. F., Workman, S. D., Boothroyd, J. C., Boulanger, M. J., Boyle, J. P. 2018; 108 (5): 519–35

  Abstract

  The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C-terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C-terminal helix are required for MAF1-mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co-immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA-competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus.

  View details for PubMedID 29505111

• Mitochondria Restrict Growth of the Intracellular Parasite Toxoplasma gondii by Limiting Its Uptake of Fatty Acids CELL METABOLISM Pernas, L., Bean, C., Boothroyd, J. C., Scorrano, L. 2018; 27 (4): 886-+

  Abstract

  How intracellular pathogens acquire essential non-diffusible host metabolites and whether the host cell counteracts the siphoning of these nutrients by its invaders are open questions. Here we show that host mitochondria fuse during infection by the intracellular parasite Toxoplasma gondii to limit its uptake of fatty acids (FAs). A combination of genetics and imaging of FA trafficking indicates that Toxoplasma infection triggers lipophagy, the autophagy of host lipid droplets (LDs), to secure cellular FAs essential for its proliferation. Indeed, Toxoplasma FA siphoning and growth are reduced in host cells genetically deficient for autophagy or triglyceride depots. Conversely, Toxoplasma FA uptake and proliferation are increased in host cells lacking mitochondrial fusion, required for efficient mitochondrial FA oxidation, or where mitochondrial FA oxidation is pharmacologically inhibited. Thus, mitochondrial fusion can be regarded as a cellular defense mechanism against intracellular parasites, by limiting Toxoplasma access to host nutrients liberated by lipophagy.

  View details for PubMedID 29617646

• MYR1-Dependent Effectors Are the Major Drivers of a Host Cell's Early Response to Toxoplasma, Including Counteracting MYR1-Independent Effects MBIO Naor, A., Panas, M. W., Marino, N., Coffey, M. J., Tonkin, C. J., Boothroyd, J. C. 2018; 9 (2)

  Abstract

  The obligate intracellular parasite Toxoplasma gondii controls its host cell from within the parasitophorous vacuole (PV) by using a number of diverse effector proteins, a subset of which require the aspartyl protease 5 enzyme (ASP5) and/or the recently discovered MYR1 protein to cross the PV membrane. To examine the impact these effectors have in the context of the entirety of the host response to Toxoplasma, we used RNA-Seq to analyze the transcriptome expression profiles of human foreskin fibroblasts infected with wild-type RH (RH-WT), RHΔmyr1, and RHΔasp5 tachyzoites. Interestingly, the majority of the differentially regulated genes responding to Toxoplasma infection are MYR1 dependent. A subset of MYR1 responses were ASP5 independent, and MYR1 function did not require ASP5 cleavage, suggesting the export of some effectors requires only MYR1. Gene set enrichment analysis of MYR1-dependent host responses suggests an upregulation of E2F transcription factors and the cell cycle and a downregulation related to interferon signaling, among numerous others. Most surprisingly, "hidden" responses arising in RHΔmyr1- but not RH-WT-infected host cells indicate counterbalancing actions of MYR1-dependent and -independent activities. The host genes and gene sets revealed here to be MYR1 dependent provide new insight into the parasite's ability to co-opt host cell functions.IMPORTANCEToxoplasma gondii is unique in its ability to successfully invade and replicate in a broad range of host species and cells within those hosts. The complex interplay of effector proteins exported by Toxoplasma is key to its success in co-opting the host cell to create a favorable replicative niche. Here we show that a majority of the transcriptomic effects in tachyzoite-infected cells depend on the activity of a novel translocation system involving MYR1 and that the effectors delivered by this system are part of an intricate interplay of activators and suppressors. Removal of all MYR1-dependent effectors reveals previously unknown activities that are masked or hidden by the action of these proteins.

  View details for PubMedID 29615509

• Toxoplasma gondii infection triggers chronic cachexia and sustained commensal dysbiosis in mice. PloS one Hatter, J. A., Kouche, Y. M., Melchor, S. J., Ng, K., Bouley, D. M., Boothroyd, J. C., Ewald, S. E. 2018; 13 (10): e0204895

  Abstract

  Toxoplasma gondii is a protozoan parasite with a predation-mediated transmission cycle between rodents and felines. Intermediate hosts acquire Toxoplasma by eating parasite cysts which invade the small intestine, disseminate systemically and finally establish host life-long chronic infection in brain and muscles. Here we show that Toxoplasma infection can trigger a severe form of sustained cachexia: a disease of progressive lean weight loss that is a causal predictor of mortality in cancer, chronic disease and many infections. Toxoplasma cachexia is characterized by acute anorexia, systemic inflammation and loss of 20% body mass. Although mice recover from symptoms of peak sickness, they fail to regain muscle mass or visceral adipose depots. We asked whether the damage to the intestinal microenvironment observed at acute time points was sustained in chronic infection and could thereby play a role in sustaining cachexia. We found that parasites replicate in the same region of the distal jejunum/proximal ileum throughout acute infection, inducing the development of secondary lymphoid structures and severe, regional inflammation. Small intestine pathology was resolved by 5 weeks post-infection. However, changes in the commensal populations, notably an outgrowth of Clostridia spp., were sustained in chronic infection. Importantly, uninfected animals co-housed with infected mice display similar changes in commensal microflora but never display symptoms of cachexia, indicating that altered commensals are not sufficient to explain the cachexia phenotype alone. These studies indicate that Toxoplasma infection is a novel and robust model to study the immune-metabolic interactions that contribute to chronic cachexia development, pathology and potential reversal.

  View details for PubMedID 30379866

• mRNA pseudouridylation affects RNA metabolism in the parasite Toxoplasma gondii RNA Nakamoto, M. A., Lovejoy, A. F., Cygan, A. M., Boothroyd, J. C. 2017; 23 (12): 1834–49

  Abstract

  RNA contains over 100 modified nucleotides that are created post-transcriptionally, among which pseudouridine (Ψ) is one of the most abundant. Although it was one of the first modifications discovered, the biological role of this modification is still not fully understood. Recently, we reported that a pseudouridine synthase (TgPUS1) is necessary for differentiation of the single-celled eukaryotic parasite Toxoplasma gondii from active to chronic infection. To better understand the biological role of pseudouridylation, we report here gel-based and deep-sequencing methods to identify TgPUS1-dependent Ψ's in Toxoplasma RNA, and the use of TgPUS1 mutants to examine the effect of this modification on mRNAs. In addition to identifying conserved sites of pseudouridylation in Toxoplasma rRNA, tRNA, and snRNA, we also report extensive pseudouridylation of Toxoplasma mRNAs, with the Ψ's being relatively depleted in the 3'-UTR but enriched at position 1 of codons. We show that many Ψ's in tRNA and mRNA are dependent on the action of TgPUS1 and that TgPUS1-dependent mRNA Ψ's are enriched in developmentally regulated transcripts. RNA-seq data obtained from wild-type and TgPUS1-mutant parasites shows that genes containing a TgPUS1-dependent Ψ are relatively more abundant in mutant parasites, while pulse/chase labeling of RNA with 4-thiouracil shows that mRNAs containing TgPUS1-dependent Ψ have a modest but statistically significant increase in half-life in the mutant parasites. These data are some of the first evidence suggesting that mRNA Ψ's play an important biological role.

  View details for PubMedID 28851751

  View details for PubMedCentralID PMC5689004

• MAF1b Binds the Host Cell MIB Complex To Mediate Mitochondrial Association. mSphere Kelly, F. D., Wei, B. M., Cygan, A. M., Parker, M. L., Boulanger, M. J., Boothroyd, J. C. 2017; 2 (3)

  Abstract

  Many diverse intracellular pathogens, such as Legionella pneumophila, Chlamydia psittaci, Encephalitozoon sp., and Toxoplasma gondii, manipulate and relocate host cell organelles, including mitochondria. Toxoplasma tachyzoites use a secreted protein, mitochondrial association factor 1b (MAF1b), to drive the association between the host mitochondria and the membrane of the parasitophorous vacuole, in which the parasites grow. The identity of the host partner in this interaction, however, has not previously been identified. By exogenously expressing tagged MAF1b in mouse embryonic fibroblasts, we were able to isolate host cell proteins that specifically interact with MAF1b. We then verified these interactions in the MAF1b-expressing fibroblasts, as well as in the context of parasite infection in human fibroblasts and HeLa cells. The results show that a host cell mitochondrial complex, the mitochondrial intermembrane space bridging (MIB) complex, specifically interacts with MAF1b. We further demonstrate that a version of MAF1b that is deficient in host-mitochondrial association does not efficiently coprecipitate the MIB complex. Validation of the importance of the MAF1b-MIB interaction came from showing that knockdown of two MIB complex components, MIC60 and SAM50, substantially reduces mitochondrial association with the parasitophorous vacuole membrane. This interaction between a secreted membrane-integral parasite protein and a membrane-bound complex of a host organelle represents the first instance of organelle relocalization in which both the host and pathogen molecules are known and provides the foundation for more detailed biochemical studies. IMPORTANCE Parasites interact intimately with their hosts, and the interactions shape both parties. The common human parasite Toxoplasma gondii replicates exclusively in a vacuole in a host cell and alters its host cell's environment through secreted proteins. One of these secreted proteins, MAF1b, acts to concentrate mitochondria around the parasite's vacuole, and this relocalization alters the host immune response. Many other intracellular pathogens also recruit host mitochondria, but the identities of the partners that mediate this interaction have not previously been described in any infection. Here, we show that Toxoplasma MAF1b binds to the multifunctional MIB protein complex on the host mitochondria. Reducing the levels of the proteins in this mitochondrial complex reduces the close association of host cell mitochondria and the parasite's vacuole. This work provides new insight into a key host-pathogen interaction and identifies possible targets for future therapeutic intervention as well as a more molecular understanding of important biology.

  View details for DOI 10.1128/mSphere.00183-17

  View details for PubMedID 28567444

• An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-kappa B-like signature in infected host cells PLOS ONE Guiton, P. S., Sagawa, J. M., Fritz, H. M., Boothroyd, J. C. 2017; 12 (3)

  Abstract

  Toxoplasmosis is a zoonotic infection affecting approximately 30% of the world's human population. After sexual reproduction in the definitive feline host, Toxoplasma oocysts, each containing 8 sporozoites, are shed into the environment where they can go on to infect humans and other warm-blooded intermediate hosts. Here, we use an in vitro model to assess host transcriptomic changes that occur in the earliest stages of such infections. We show that infection of rat intestinal epithelial cells with mature sporozoites primarily results in higher expression of genes associated with Tumor Necrosis Factor alpha (TNFα) signaling via NF-κB. Furthermore, we find that, consistent with their biology, these mature, invaded sporozoites display a transcriptome intermediate between the previously reported day 10 oocysts and that of their tachyzoite counterparts. Thus, this study uncovers novel host and pathogen factors that may be critical for the establishment of a successful intracellular niche following sporozoite-initiated infection.

  View details for DOI 10.1371/journal.pone.0173018

  View details for Web of Science ID 000399175000005

  View details for PubMedID 28362800

• Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1. mBio Child, M. A., Garland, M., Foe, I., Madzelan, P., Treeck, M., van der Linden, W. A., Oresic Bender, K., Weerapana, E., Wilson, M. A., Boothroyd, J. C., Reese, M. L., Bogyo, M. 2017; 8 (1)

  Abstract

  Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

  View details for DOI 10.1128/mBio.02189-16

  View details for PubMedID 28246362

• Toxoplasma growth in vitro is dependent on exogenous tyrosine and is independent of AAH2 even in tyrosine-limiting conditions. Experimental parasitology Marino, N. D., Boothroyd, J. C. 2017

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite capable of infecting virtually all nucleated cell types in almost all warm-blooded animals. Interestingly, Toxoplasma has a relatively full repertoire of amino acid biosynthetic machinery, perhaps reflecting its broad host range and, consequently, its need to adapt to a wide array of amino acid resources. Although Toxoplasma has been shown to be auxotrophic for tryptophan and arginine, it has not previously been determined if Toxoplasma is also auxotrophic for tyrosine. Toxoplasma tachyzoites and bradyzoites were recently found to express an amino acid hydroxylase (AAH2) that is capable of synthesizing tyrosine and dihydroxyphenylalanine (DOPA) from phenylalanine; however, the role of AAH2 in tachyzoite and bradyzoite infection has not yet been identified. To determine if Toxoplasma requires exogenous tyrosine for growth, we performed growth assays on tachyzoites and bradyzoites in nutrient-rich media titrated with varying amounts of tyrosine. We found that Toxoplasma tachyzoites form significantly smaller plaques in tyrosine-limiting media in a dose-dependent manner and that this phenotype is not affected by deletion of TgAAH2. To determine if bradyzoites require exogenous tyrosine for growth, we induced differentiation from tachyzoites in vitro in tyrosine-limiting media and found that replication and vacuole number are all decreased in tyrosine-deficient media. Importantly, culture of confluent human fibroblasts in tyrosine-deficient media does not affect their viability, indicating that, at least in vitro, the need for tyrosine is at the level of Toxoplasma, not the host cell supporting its growth.

  View details for DOI 10.1016/j.exppara.2017.02.018

  View details for PubMedID 28257757

  View details for PubMedCentralID PMC5423395

• Toxoplasma DJ-1 Regulates Organelle Secretion by a Direct Interaction with Calcium-Dependent Protein Kinase 1 MBIO Child, M. A., Garland, M., Foe, I., Madzelan, P., Treeck, M., van der Linden, W. A., Bender, K. O., Weerapana, E., Wilson, M. A., Boothroyd, J. C., Reese, M. L., Bogyo, M. 2017; 8 (1)

  Abstract

  Human DJ-1 is a highly conserved and yet functionally enigmatic protein associated with a heritable form of Parkinson's disease. It has been suggested to be a redox-dependent regulatory scaffold, binding to proteins to modulate their function. Here we present the X-ray crystal structure of the Toxoplasma orthologue Toxoplasma gondii DJ-1 (TgDJ-1) at 2.1-Å resolution and show that it directly associates with calcium-dependent protein kinase 1 (CDPK1). The TgDJ-1 structure identifies an orthologously conserved arginine dyad that acts as a phospho-gatekeeper motif to control complex formation. We determined that the binding of TgDJ-1 to CDPK1 is sensitive to oxidation and calcium, and that this interaction potentiates CDPK1 kinase activity. Finally, we show that genetic deletion of TgDJ-1 results in upregulation of CDPK1 expression and that disruption of the CDPK1/TgDJ-1 complex in vivo prevents normal exocytosis of parasite virulence-associated organelles called micronemes. Overall, our data suggest that TgDJ-1 functions as a noncanonical kinase-regulatory scaffold that integrates multiple intracellular signals to tune microneme exocytosis in T. gondiiIMPORTANCE Apicomplexan parasites such as Toxoplasma and Plasmodium are obligate intracellular parasites that require the protective environment of a host cell in order to replicate and survive within a host organism. These parasites secrete effector proteins from specialized apical organelles to select and invade a chosen host cell. The secretion of these organelles is a tightly regulated process coordinated by endogenous small molecules and calcium-dependent protein kinases. We previously identified the Toxoplasma orthologue of the highly conserved protein DJ-1 as a regulator of microneme secretion, but the molecular basis for this was not known. We have now identified the molecular mechanism for how TgDJ-1 regulates microneme secretion. TgDJ-1 interacts with the kinase responsible for the secretion of these organelles (calcium-dependent kinase 1) and synergizes with calcium to potentiate kinase activity. This interaction is direct, phosphodependent, and necessary for the normal secretion of these important organelles.

  View details for DOI 10.1128/mBio.02189-16

  View details for Web of Science ID 000395835000009

  View details for PubMedCentralID PMC5347346

• A Novel Secreted Protein, MYR1, Is Central to Toxoplasma's Manipulation of Host Cells. mBio Franco, M., Panas, M. W., Marino, N. D., Lee, M. W., Buchholz, K. R., Kelly, F. D., Bednarski, J. J., Sleckman, B. P., Pourmand, N., Boothroyd, J. C. 2016; 7 (1)

  Abstract

  The intracellular protozoan Toxoplasma gondii dramatically reprograms the transcriptome of host cells it infects, including substantially up-regulating the host oncogene c-myc. By applying a flow cytometry-based selection to infected mouse cells expressing green fluorescent protein fused to c-Myc (c-Myc-GFP), we isolated mutant tachyzoites defective in this host c-Myc up-regulation. Whole-genome sequencing of three such mutants led to the identification of MYR1 (Myc regulation 1; TGGT1_254470) as essential for c-Myc induction. MYR1 is a secreted protein that requires TgASP5 to be cleaved into two stable portions, both of which are ultimately found within the parasitophorous vacuole and at the parasitophorous vacuole membrane. Deletion of MYR1 revealed that in addition to its requirement for c-Myc up-regulation, the MYR1 protein is needed for the ability of Toxoplasma tachyzoites to modulate several other important host pathways, including those mediated by the dense granule effectors GRA16 and GRA24. This result, combined with its location at the parasitophorous vacuole membrane, suggested that MYR1 might be a component of the machinery that translocates Toxoplasma effectors from the parasitophorous vacuole into the host cytosol. Support for this possibility was obtained by showing that transit of GRA24 to the host nucleus is indeed MYR1-dependent. As predicted by this pleiotropic phenotype, parasites deficient in MYR1 were found to be severely attenuated in a mouse model of infection. We conclude, therefore, that MYR1 is a novel protein that plays a critical role in how Toxoplasma delivers effector proteins to the infected host cell and that this is crucial to virulence.Toxoplasma gondii is an important human pathogen and a model for the study of intracellular parasitism. Infection of the host cell with Toxoplasma tachyzoites involves the introduction of protein effectors, including many that are initially secreted into the parasitophorous vacuole but must ultimately translocate to the host cell cytosol to function. The work reported here identified a novel protein that is required for this translocation. These results give new insight into a very unusual cell biology process as well as providing a potential handle on a pathway that is necessary for virulence and, therefore, a new potential target for chemotherapy.

  View details for DOI 10.1128/mBio.02231-15

  View details for PubMedID 26838724

  View details for PubMedCentralID PMC4742717

• Not a Simple Tether: Binding of Toxoplasma gondii AMA1 to RON2 during Invasion Protects AMA1 from Rhomboid-Mediated Cleavage and Leads to Dephosphorylation of Its Cytosolic Tail. mBio Krishnamurthy, S., Deng, B., del Rio, R., Buchholz, K. R., Treeck, M., Urban, S., Boothroyd, J., Lam, Y., Ward, G. E. 2016; 7 (5)

  Abstract

  Apical membrane antigen 1 (AMA1) is a receptor protein on the surface of Toxoplasma gondii that plays a critical role in host cell invasion. The ligand to which T gondii AMA1 (TgAMA1) binds, TgRON2, is secreted into the host cell membrane by the parasite during the early stages of invasion. The TgAMA1-TgRON2 complex forms the core of the "moving junction," a ring-shaped zone of tight contact between the parasite and host cell membranes, through which the parasite pushes itself during invasion. Paradoxically, the parasite also expresses rhomboid proteases that constitutively cleave the TgAMA1 transmembrane domain. How can TgAMA1 function effectively in host cell binding if its extracellular domain is constantly shed from the parasite surface? We show here that when TgAMA1 binds the domain 3 (D3) peptide of TgRON2, its susceptibility to cleavage by rhomboid protease(s) is greatly reduced. This likely serves to maintain parasite-host cell binding at the moving junction, a hypothesis supported by data showing that parasites expressing a hypercleavable version of TgAMA1 invade less efficiently than wild-type parasites do. Treatment of parasites with the D3 peptide was also found to reduce phosphorylation of S527 on the cytoplasmic tail of TgAMA1, and parasites expressing a phosphomimetic S527D allele of TgAMA1 showed an invasion defect. Taken together, these data suggest that TgAMA1-TgRON2 interaction at the moving junction protects TgAMA1 molecules that are actively engaged in host cell penetration from rhomboid-mediated cleavage and generates an outside-in signal that leads to dephosphorylation of the TgAMA1 cytosolic tail. Both of these effects are required for maximally efficient host cell invasion.Nearly one-third of the world's population is infected with the protozoan parasite Toxoplasma gondii, which causes life-threatening disease in neonates and immunocompromised individuals. T. gondii is a member of the phylum Apicomplexa, which includes many other parasites of veterinary and medical importance, such as those that cause coccidiosis, babesiosis, and malaria. Apicomplexan parasites grow within their hosts through repeated cycles of host cell invasion, parasite replication, and host cell lysis. Parasites that cannot invade host cells cannot survive or cause disease. AMA1 is a highly conserved protein on the surface of apicomplexan parasites that is known to be important for invasion, and the work presented here reveals new and unexpected insights into AMA1 function. A more complete understanding of the role of AMA1 in invasion may ultimately contribute to the development of new chemotherapeutics designed to disrupt AMA1 function and invasion-related signaling in this important group of human pathogens.

  View details for DOI 10.1128/mBio.00754-16

  View details for PubMedID 27624124

• Local admixture of amplified and diversified secreted pathogenesis determinants shapes mosaic Toxoplasma gondii genomes. Nature communications Lorenzi, H., Khan, A., Behnke, M. S., Namasivayam, S., Swapna, L. S., Hadjithomas, M., Karamycheva, S., Pinney, D., Brunk, B. P., Ajioka, J. W., Ajzenberg, D., Boothroyd, J. C., Boyle, J. P., Dardé, M. L., Diaz-Miranda, M. A., Dubey, J. P., Fritz, H. M., Gennari, S. M., Gregory, B. D., Kim, K., Saeij, J. P., Su, C., White, M. W., Zhu, X., Howe, D. K., Rosenthal, B. M., Grigg, M. E., Parkinson, J., Liu, L., Kissinger, J. C., Roos, D. S., David Sibley, L. 2016; 7: 10147-?

  Abstract

  Toxoplasma gondii is among the most prevalent parasites worldwide, infecting many wild and domestic animals and causing zoonotic infections in humans. T. gondii differs substantially in its broad distribution from closely related parasites that typically have narrow, specialized host ranges. To elucidate the genetic basis for these differences, we compared the genomes of 62 globally distributed T. gondii isolates to several closely related coccidian parasites. Our findings reveal that tandem amplification and diversification of secretory pathogenesis determinants is the primary feature that distinguishes the closely related genomes of these biologically diverse parasites. We further show that the unusual population structure of T. gondii is characterized by clade-specific inheritance of large conserved haploblocks that are significantly enriched in tandemly clustered secretory pathogenesis determinants. The shared inheritance of these conserved haploblocks, which show a different ancestry than the genome as a whole, may thus influence transmission, host range and pathogenicity.

  View details for DOI 10.1038/ncomms10147

  View details for PubMedID 26738725

• An aspartyl protease defines a novel pathway for export of Toxoplasma proteins into the host cell ELIFE Coffey, M. J., Sleebs, B. E., Uboldi, A. D., Garnham, A., Franco, M., Marino, N. D., Panas, M. W., Ferguson, D. J., Enciso, M., O'Neill, M. T., Lopaticki, S., Stewart, R. J., Dewson, G., Smyth, G. K., Smith, B. J., Masters, S. L., Boothroyd, J. C., Boddey, J. A., Tonkin, C. J. 2015; 4

  Abstract

  Infection by Toxoplasma gondii leads to massive changes to the host cell. Here, we identify a novel host cell effector export pathway that requires the Golgi-resident aspartyl protease 5 (ASP5). We demonstrate that ASP5 cleaves a highly constrained amino acid motif that has similarity to the PEXEL-motif of Plasmodium parasites. We show that ASP5 matures substrates at both the N- and C-terminal ends of proteins and also controls trafficking of effectors without this motif. Furthermore, ASP5 controls establishment of the nanotubular network and is required for the efficient recruitment of host mitochondria to the vacuole. Assessment of host gene expression reveals that the ASP5-dependent pathway influences thousands of the transcriptional changes that Toxoplasma imparts on its host cell. All these changes result in attenuation of virulence of Δasp5 tachyzoites in vivo. This work characterizes the first identified machinery required for export of Toxoplasma effectors into the infected host cell.

  View details for DOI 10.7554/eLife.10809

  View details for Web of Science ID 000373852900001

  View details for PubMedID 26576949

  View details for PubMedCentralID PMC4764566

• Internalization and TLR-dependent type I interferon production by monocytes in response to Toxoplasma gondii IMMUNOLOGY AND CELL BIOLOGY Han, S., Melichar, H. J., Coombes, J. L., Chan, S. W., Koshy, A. A., Boothroyd, J. C., Barton, G. M., Robey, E. A. 2014; 92 (10): 872-881

  Abstract

  The classic anti-viral cytokine interferon (IFN)-β can be induced during parasitic infection, but relatively little is know about the cell types and signaling pathways involved. Here we show that inflammatory monocytes (IMs), but not neutrophils, produce IFN-β in response to T. gondii infection. This difference correlated with the mode of parasite entry into host cells, with phagocytic uptake predominating in IMs and active invasion predominating in neutrophils. We also show that expression of IFN-β requires phagocytic uptake of the parasite by IMs, and signaling through Toll-like receptors (TLRs) and MyD88. Finally, we show that IMs are major producers of IFN-β in mesenteric lymph nodes following in vivo oral infection of mice, and mice lacking the receptor for type I IFN-1 show higher parasite loads and reduced survival. Our data reveal a TLR and internalization-dependent pathway in IMs for IFN-β induction to a non-viral pathogen.

  View details for DOI 10.1038/icb.2014.70

  View details for Web of Science ID 000345496900008

  View details for PubMedID 25155465

  View details for PubMedCentralID PMC4245188

• The Toxoplasma Pseudokinase ROP5 Is an Allosteric Inhibitor of the Immunity-related GTPases JOURNAL OF BIOLOGICAL CHEMISTRY Reese, M. L., Shah, N., Boothroyd, J. C. 2014; 289 (40): 27849-27858

  Abstract

  The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4-10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.

  View details for DOI 10.1074/jbc.M114.567057

  View details for Web of Science ID 000342852800041

  View details for PubMedID 25118287

  View details for PubMedCentralID PMC4183819

• Use of Transgenic Parasites and Host Reporters To Dissect Events That Promote Interleukin-12 Production during Toxoplasmosis INFECTION AND IMMUNITY Christian, D. A., Koshy, A. A., Reuter, M. A., Betts, M. R., Boothroyd, J. C., Hunter, C. A. 2014; 82 (10): 4056-4067

  Abstract

  The intracellular parasite Toxoplasma gondii has multiple strategies to alter host cell function, including the injection of rhoptry proteins into the cytosol of host cells as well as bystander populations, but the consequence of these events is unclear. Here, a reporter system using fluorescent parasite strains that inject Cre recombinase with their rhoptry proteins (Toxoplasma-Cre) was combined with Ai6 Cre reporter mice to identify cells that have been productively infected, that have been rhoptry injected but lack the parasite, or that have phagocytosed T. gondii. The ability to distinguish these host-parasite interactions was then utilized to dissect the events that lead to the production of interleukin-12 p40 (IL-12p40), which is required for resistance to T. gondii. In vivo, the use of invasion-competent or invasion-inhibited (phagocytosed) parasites with IL-12p40 (YET40) reporter mice revealed that dendritic cell (DC) and macrophage populations that phagocytose the parasite or are infected can express IL-12p40 but are not the major source, as larger numbers of uninfected cells secrete this cytokine. Similarly, the use of Toxoplasma-Cre parasite strains indicated that dendritic cells and inflammatory monocytes untouched by the parasite and not cells injected by the parasite are the primary source of IL-12p40. These results imply that a soluble host or parasite factor is responsible for the bulk of IL-12p40 production in vivo, rather than cellular interactions with T. gondii that result in infection, infection and clearance, injection of rhoptry proteins, or phagocytosis of the parasite.

  View details for DOI 10.1128/IAI.01643-14

  View details for Web of Science ID 000341935100007

  View details for PubMedID 25024368

  View details for PubMedCentralID PMC4187868

• Immune Profiling of Pregnant Toxoplasma-Infected US and Colombia Patients Reveals Surprising Impacts of Infection on Peripheral Blood Cytokines. journal of infectious diseases Pernas, L., Ramirez, R., Holmes, T. H., Montoya, J. G., Boothroyd, J. C. 2014; 210 (6): 923-931

  Abstract

  In North America (NA) and Europe, the majority of toxoplasmosis cases are benign and generally asymptomatic, while in South America (SA) toxoplasmosis is associated with much more severe symptoms in adults and congenitally infected children. The reasons for these differences remain unknown and, currently, there is little information from patients in either region on how the immune system responds to infection with Toxoplasma gondii. Here, we report the relative abundance of 51 serum cytokines from acute and chronic toxoplasmosis cohorts of pregnant women from the United States (US), where approximately one-half of clinical isolates are Type II, and Colombia, where clinical isolates are generally "atypical" or Type I-like strains. Surprisingly, the results showed notably lower levels of 23 cytokines in acutely infected patients from the US, relative to uninfected US controls. In acutely infected Colombian patients, however, only 8 cytokine levels differed detectably with four being lower and four higher relative to uninfected controls. Strikingly, there were also differences in the cytokine profiles of the chronically infected patients relative to uninfected controls in the US cohort. Hence, Toxoplasma appears to specifically impact levels of circulating cytokines and our results may partly explain region-specific differences in the clinical spectrum of toxoplasmosis.

  View details for DOI 10.1093/infdis/jiu189

  View details for PubMedID 24664173

• Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses CELL REPORTS Grover, H. S., Chu, H. H., Kelly, F. D., Yang, S. J., Reese, M. L., Blanchard, N., Gonzalez, F., Chan, S. W., Boothroyd, J. C., Shastri, N., Robey, E. A. 2014; 7 (5): 1716-1728

  Abstract

  CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.

  View details for DOI 10.1016/j.celrep.2014.04.031

  View details for Web of Science ID 000338324200034

  View details for PubMedCentralID PMC4057976

• Impact of Regulated Secretion on Antiparasitic CD8 T Cell Responses. Cell reports Grover, H. S., Chu, H. H., Kelly, F. D., Yang, S. J., Reese, M. L., Blanchard, N., Gonzalez, F., Chan, S. W., Boothroyd, J. C., Shastri, N., Robey, E. A. 2014; 7 (5): 1716-28

  Abstract

  CD8 T cells play a key role in defense against the intracellular parasite Toxoplasma, but why certain CD8 responses are more potent than others is not well understood. Here, we describe a parasite antigen, ROP5, that elicits a CD8 T cell response in genetically susceptible mice. ROP5 is secreted via parasite organelles termed rhoptries that are injected directly into host cells during invasion, whereas the protective, dense-granule antigen GRA6 is constitutively secreted into the parasitophorous vacuole. Transgenic parasites in which the ROP5 antigenic epitope was targeted for secretion through dense granules led to enhanced CD8 T cell responses, whereas targeting the GRA6 epitope to rhoptries led to reduced CD8 responses. CD8 T cell responses to the dense-granule-targeted ROP5 epitope resulted in reduced parasite load in the brain. These data suggest that the mode of secretion affects the efficacy of parasite-specific CD8 T cell responses.

  View details for DOI 10.1016/j.celrep.2014.04.031

  View details for PubMedID 24857659

  View details for PubMedCentralID PMC4057976

• GRA25 Is a Novel Virulence Factor of Toxoplasma gondii and Influences the Host Immune Response. Infection and immunity Shastri, A. J., Marino, N. D., Franco, M., Lodoen, M. B., Boothroyd, J. C. 2014; 82 (6): 2595-2605

  Abstract

  The obligate intracellular parasite Toxoplasma gondii is able to infect a broad range of hosts and cell types due, in part, to the diverse arsenal of effectors it secretes into the host cell. Here, using genetic crosses between Type II and Type III Toxoplasma strains and QTL mapping of the changes they induce in macrophage gene expression, we identify a novel dense granule protein, GRA25. Encoded on chromosome IX, GRA25 is a phosphoprotein that is secreted outside of the parasites and is found within the parasitophorous vacuole. In vitro experiments with a Type II Δgra25 strain showed that macrophages infected with this strain secrete lower levels of CCL2 and CXCL1 compared to those infected with the wild type or complemented control parasites. In vivo experiments showed that mice infected with a Type II Δgra25 strain are able to survive an otherwise lethal dose of Toxoplasma tachyzoites, and complementation of the mutant with an ectopic copy of GRA25 largely rescues this phenotype. Interestingly, the Type II and Type III versions of GRA25 vary in endogenous expression; however, both are able to promote parasite expansion in vivo when expressed in a Type II Δgra25 strain. These data establish GRA25 as a novel virulence factor and immune modulator.

  View details for DOI 10.1128/IAI.01339-13

  View details for PubMedID 24711568

• The calcium-dependent protein kinase 3 of toxoplasma influences basal calcium levels and functions beyond egress as revealed by quantitative phosphoproteome analysis. PLoS pathogens Treeck, M., Sanders, J. L., Gaji, R. Y., LaFavers, K. A., Child, M. A., Arrizabalaga, G., Elias, J. E., Boothroyd, J. C. 2014; 10 (6)

  Abstract

  Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  View details for DOI 10.1371/journal.ppat.1004197

  View details for PubMedID 24945436

• The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis PLOS PATHOGENS Treeck, M., Sanders, J. L., Gaji, R. Y., LaFavers, K. A., Child, M. A., Arrizabalaga, G., Elias, J. E., Boothroyd, J. C. 2014; 10 (6)

  Abstract

  Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.

  View details for DOI 10.1371/journal.ppat.1004197

  View details for Web of Science ID 000338197400032

  View details for PubMedCentralID PMC4063958

• Infection by Toxoplasma gondii specifically induces host c-Myc and the genes this pivotal transcription factor regulates. Eukaryotic cell Franco, M., Shastri, A. J., Boothroyd, J. C. 2014; 13 (4): 483-493

  Abstract

  Toxoplasma gondii infection has previously been described to cause dramatic changes in the host transcriptome by manipulating key regulators, including STATs, NF-κB, and microRNAs. Here, we report that Toxoplasma tachyzoites also mediate rapid and sustained induction of another pivotal regulator of host cell transcription, c-Myc. This induction is seen in cells infected with all three canonical types of Toxoplasma but not the closely related apicomplexan parasite Neospora caninum. Coinfection of cells with both Toxoplasma and Neospora still results in an increase in the level of host c-Myc, showing that c-Myc is actively upregulated by Toxoplasma infection (rather than repressed by Neospora). We further demonstrate that this upregulation may be mediated through c-Jun N-terminal protein kinase (JNK) and is unlikely to be a nonspecific host response, as heat-killed Toxoplasma parasites do not induce this increase and neither do nonviable parasites inside the host cell. Finally, we show that the induced c-Myc is active and that transcripts dependent on its function are upregulated, as predicted. Hence, c-Myc represents an additional way in which Toxoplasma tachyzoites have evolved to specifically alter host cell functions during intracellular growth.

  View details for DOI 10.1128/EC.00316-13

  View details for PubMedID 24532536

• Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response. PLoS biology Pernas, L., Adomako-Ankomah, Y., Shastri, A. J., Ewald, S. E., Treeck, M., Boyle, J. P., Boothroyd, J. C. 2014; 12 (4)

  Abstract

  Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria.

  View details for DOI 10.1371/journal.pbio.1001845

  View details for PubMedID 24781109

• NLRP1 Is an Inflammasome Sensor for Toxoplasma gondii INFECTION AND IMMUNITY Ewald, S. E., Chavarria-Smith, J., Boothroyd, J. C. 2014; 82 (1): 460-468

  Abstract

  The obligate intracellular parasite Toxoplasma gondii is able to infect nearly all nucleated cell types of warm-blooded animals. This is achieved through the injection of hundreds of parasite effectors into the host cell cytosol, allowing the parasite to establish a vacuolar niche for growth, replication, and persistence. Here we show that Toxoplasma infection actives an inflammasome response in mice and rats, an innate immune sensing system designed to survey the host cytosol for foreign components leading to inflammation and cell death. Oral infection with Toxoplasma triggers an inflammasome response that is protective to the host, limiting parasite load and dissemination. Toxoplasma infection is sufficient to generate an inflammasome response in germfree animals. Interleukin 1β (IL-1β) secretion by macrophage requires the effector caspases 1 and 11, the adapter ASC, and NLRP1, the sensor previously described to initiate the inflammasome response to Bacillus anthracis lethal factor. The allele of NLRP1b derived from 129 mice is sufficient to enhance the B6 bone marrow-derived macrophage (BMDM) inflammasome response to Toxoplasma independent of the lethal factor proteolysis site. Moreover, N-terminal processing of NLRP1b, the only mechanism of activation known to date, is not observed in response to Toxoplasma infection. Cumulatively, these data indicate that NLRP1 is an innate immune sensor for Toxoplasma infection, activated via a novel mechanism that corresponds to a host-protective innate immune response to the parasite.

  View details for DOI 10.1128/IAI.01170-13

  View details for Web of Science ID 000328899600047

  View details for PubMedID 24218483

  View details for PubMedCentralID PMC3911858

• Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion. Nature chemical biology Child, M. A., Hall, C. I., Beck, J. R., Ofori, L. O., Albrow, V. E., Garland, M., Bowyer, P. W., Bradley, P. J., Powers, J. C., Boothroyd, J. C., Weerapana, E., Bogyo, M. 2013; 9 (10): 651-656

  Abstract

  Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

  View details for DOI 10.1038/nchembio.1315

  View details for PubMedID 23934245

• A nucleotide sugar transporter involved in glycosylation of the toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS pathogens Caffaro, C. E., Koshy, A. A., Liu, L., Zeiner, G. M., Hirschberg, C. B., Boothroyd, J. C. 2013; 9 (5)

  View details for DOI 10.1371/journal.ppat.1003331

  View details for PubMedID 23658519

• Have it your way: how polymorphic, injected kinases and pseudokinases enable toxoplasma to subvert host defenses. PLoS pathogens Boothroyd, J. C. 2013; 9 (4)

  View details for DOI 10.1371/journal.ppat.1003296

  View details for PubMedID 23633947

  View details for PubMedCentralID PMC3635977

• Bradyzoite Pseudokinase 1 Is Crucial for Efficient Oral Infectivity of the Toxoplasma gondii Tissue Cyst EUKARYOTIC CELL Buchholz, K. R., Bowyer, P. W., Boothroyd, J. C. 2013; 12 (3): 399-410

  View details for DOI 10.1128/EC.00343-12

  View details for Web of Science ID 000315405800003

  View details for PubMedID 23291621

• A nucleotide sugar transporter involved in glycosylation of the Toxoplasma tissue cyst wall is required for efficient persistence of bradyzoites. PLoS pathogens Caffaro, C. E., Koshy, A. A., Liu, L., Zeiner, G. M., Hirschberg, C. B., Boothroyd, J. C. 2013; 9 (5)

  Abstract

  Toxoplasma gondii is an intracellular parasite that transitions from acute infection to a chronic infective state in its intermediate host via encystation, which enables the parasite to evade immune detection and clearance. It is widely accepted that the tissue cyst perimeter is highly and specifically decorated with glycan modifications; however, the role of these modifications in the establishment and persistence of chronic infection has not been investigated. Here we identify and biochemically and biologically characterize a Toxoplasma nucleotide-sugar transporter (TgNST1) that is required for cyst wall glycosylation. Toxoplasma strains deleted for the TgNST1 gene (Δnst1) form cyst-like structures in vitro but no longer interact with lectins, suggesting that Δnst1 strains are deficient in the transport and use of sugars for the biosynthesis of cyst-wall structures. In vivo infection experiments demonstrate that the lack of TgNST1 activity does not detectably impact the acute (tachyzoite) stages of an infection or tropism of the parasite for the brain but that Δnst1 parasites are severely defective in persistence during the chronic stages of the infection. These results demonstrate for the first time the critical role of parasite glycoconjugates in the persistence of Toxoplasma tissue cysts.

  View details for DOI 10.1371/journal.ppat.1003331

  View details for PubMedID 23658519

  View details for PubMedCentralID PMC3642066

• Toxoplasma gondii Sporozoites Invade Host Cells Using Two Novel Paralogues of RON2 and AMA1. PloS one Poukchanski, A., Fritz, H. M., Tonkin, M. L., Treeck, M., Boulanger, M. J., Boothroyd, J. C. 2013; 8 (8)

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.

  View details for DOI 10.1371/journal.pone.0070637

  View details for PubMedID 23940612

  View details for PubMedCentralID PMC3734201

• A Forward Genetic Screen Reveals that Calcium-dependent Protein Kinase 3 Regulates Egress in Toxoplasma PLOS PATHOGENS Garrison, E., Treeck, M., Ehret, E., Butz, H., Garbuz, T., Oswald, B. P., Settles, M., Boothroyd, J., Arrizabalaga, G. 2012; 8 (11)

  Abstract

  Egress from the host cell is a crucial and highly regulated step in the biology of the obligate intracellular parasite, Toxoplasma gondii. Active egress depends on calcium fluxes and appears to be a crucial step in escaping the attack from the immune system and, potentially, in enabling the parasites to shuttle into appropriate cells for entry into the brain of the host. Previous genetic screens have yielded mutants defective in both ionophore-induced egress and ionophore-induced death. Using whole genome sequencing of one mutant and subsequent analysis of all mutants from these screens, we find that, remarkably, four independent mutants harbor a mis-sense mutation in the same gene, TgCDPK3, encoding a calcium-dependent protein kinase. All four mutations are predicted to alter key regions of TgCDPK3 and this is confirmed by biochemical studies of recombinant forms of each. By complementation we confirm a crucial role for TgCDPK3 in the rapid induction of parasite egress and we establish that TgCDPK3 is critical for formation of latent stages in the brains of mice. Genetic knockout of TgCDPK3 confirms a crucial role for this kinase in parasite egress and a non-essential role for it in the lytic cycle.

  View details for DOI 10.1371/journal.ppat.1003049

  View details for Web of Science ID 000311997100060

  View details for PubMedID 23209419

• Toxoplasma Co-opts Host Cells It Does Not Invade PLOS PATHOGENS Koshy, A. A., Dietrich, H. K., Christian, D. A., Melehani, J. H., Shastri, A. J., Hunter, C. A., Boothroyd, J. C. 2012; 8 (7)

  Abstract

  Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.

  View details for DOI 10.1371/journal.ppat.1002825

  View details for Web of Science ID 000306837700043

  View details for PubMedID 22910631

  View details for PubMedCentralID PMC3406079

• A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins PLOS BIOLOGY Fleckenstein, M. C., Reese, M. L., Koenen-Waisman, S., Boothroyd, J. C., Howard, J. C., Steinfeldt, T. 2012; 10 (7)

  Abstract

  The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.

  View details for DOI 10.1371/journal.pbio.1001358

  View details for Web of Science ID 000307161000004

  View details for PubMedID 22802726

• Infected Dendritic Cells Facilitate Systemic Dissemination and Transplacental Passage of the Obligate Intracellular Parasite Neospora caninum in Mice PLOS ONE Collantes-Fernandez, E., Arrighi, R. B., Alvarez-Garcia, G., Weidner, J. M., Regidor-Cerrillo, J., Boothroyd, J. C., Ortega-Mora, L. M., Barragan, A. 2012; 7 (3)

  Abstract

  The obligate intracellular parasite Neospora caninum disseminates across the placenta and the blood-brain barrier, to reach sites where it causes severe pathology or establishes chronic persistent infections. The mechanisms used by N. caninum to breach restrictive biological barriers remain elusive. To examine the cellular basis of these processes, migration of different N. caninum isolates (Nc-1, Nc-Liverpool, Nc-SweB1 and the Spanish isolates: Nc-Spain 3H, Nc-Spain 4H, Nc-Spain 6, Nc-Spain 7 and Nc-Spain 9) was studied in an in vitro model based on a placental trophoblast-derived BeWo cell line. Here, we describe that infection of dendritic cells (DC) by N. caninum tachyzoites potentiated translocation of parasites across polarized cellular monolayers. In addition, powered by the parasite's own gliding motility, extracellular N. caninum tachyzoites were able to transmigrate across cellular monolayers. Altogether, the presented data provides evidence of two putative complementary pathways utilized by N. caninum, in an isolate-specific fashion, for passage of restrictive cellular barriers. Interestingly, adoptive transfer of tachyzoite-infected DC in mice resulted in increased parasitic loads in various organs, e.g. the central nervous system, compared to infections with free parasites. Inoculation of pregnant mice with infected DC resulted in an accentuated vertical transmission to the offspring with increased parasitic loads and neonatal mortality. These findings reveal that N. caninum exploits the natural cell trafficking pathways in the host to cross cellular barriers and disseminate to deep tissues. The findings are indicative of conserved dissemination strategies among coccidian apicomplexan parasites.

  View details for DOI 10.1371/journal.pone.0032123

  View details for Web of Science ID 000303017700024

  View details for PubMedID 22403627

• Transcriptomic Analysis of Toxoplasma Development Reveals Many Novel Functions and Structures Specific to Sporozoites and Oocysts PLOS ONE Fritz, H. M., Buchholz, K. R., Chen, X., Durbin-Johnson, B., Rocke, D. M., Conrad, P. A., Boothroyd, J. C. 2012; 7 (2)

  Abstract

  Sexual reproduction of Toxoplasma gondii occurs exclusively within enterocytes of the definitive felid host. The resulting immature oocysts are excreted into the environment during defecation, where in the days following, they undergo a complex developmental process. Within each oocyst, this culminates in the generation of two sporocysts, each containing 4 sporozoites. A single felid host is capable of shedding millions of oocysts, which can survive for years in the environment, are resistant to most methods of microbial inactivation during water-treatment and are capable of producing infection in warm-blooded hosts at doses as low as 1-10 ingested oocysts. Despite its extremely interesting developmental biology and crucial role in initiating an infection, almost nothing is known about the oocyst stage beyond morphological descriptions. Here, we present a complete transcriptomic analysis of the oocyst from beginning to end of its development. In addition, and to identify genes whose expression is unique to this developmental form, we compared the transcriptomes of developing oocysts with those of in vitro-derived tachyzoites and in vivo-derived bradyzoites. Our results reveal many genes whose expression is specifically up- or down-regulated in different developmental stages, including many genes that are likely critical to oocyst development, wall formation, resistance to environmental destruction and sporozoite infectivity. Of special note is the up-regulation of genes that appear "off" in tachyzoites and bradyzoites but that encode homologues of proteins known to serve key functions in those asexual stages, including a novel pairing of sporozoite-specific paralogues of AMA1 and RON2, two proteins that have recently been shown to form a crucial bridge during tachyzoite invasion of host cells. This work provides the first in-depth insight into the development and functioning of one of the most important but least studied stages in the Toxoplasma life cycle.

  View details for DOI 10.1371/journal.pone.0029998

  View details for Web of Science ID 000302733900003

  View details for PubMedID 22347997

• Proteomic Analysis of Fractionated Toxoplasma Oocysts Reveals Clues to Their Environmental Resistance PLOS ONE Fritz, H. M., Bowyer, P. W., Bogyo, M., Conrad, P. A., Boothroyd, J. C. 2012; 7 (1)

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite that is unique in its ability to infect a broad range of birds and mammals, including humans, leading to an extremely high worldwide prevalence and distribution. This work focuses on the environmentally resistant oocyst, which is the product of sexual replication in felids and an important source of human infection. Due to the difficulty in producing and working with oocysts, relatively little is known about how this stage is able to resist extreme environmental stresses and how they initiate a new infection, once ingested. To fill this gap, the proteome of the wall and sporocyst/sporozoite fractions of mature, sporulated oocysts were characterized using one-dimensional gel electrophoresis followed by LC-MS/MS on trypsin-digested peptides. A combined total of 1021 non-redundant T. gondii proteins were identified in the sporocyst/sporozoite fraction and 226 were identified in the oocyst wall fraction. Significantly, 172 of the identified proteins have not previously been identified in Toxoplasma proteomic studies. Among these are several of interest for their likely role in conferring environmental resistance including a family of small, tyrosine-rich proteins present in the oocyst wall fractions and late embryogenesis abundant domain-containing (LEA) proteins in the cytosolic fractions. The latter are known from other systems to be key to enabling survival against desiccation.

  View details for DOI 10.1371/journal.pone.0029955

  View details for Web of Science ID 000299771900033

  View details for PubMedID 22279555

  View details for PubMedCentralID PMC3261165

• Tissue Barriers of the Human Placenta to Infection with Toxoplasma gondii INFECTION AND IMMUNITY Robbins, J. R., Zeldovich, V. B., Poukchanski, A., Boothroyd, J. C., Bakardjiev, A. I. 2012; 80 (1): 418-428

  Abstract

  Toxoplasma gondii is a ubiquitous, obligate intracellular parasite capable of crossing the placenta to cause spontaneous abortion, preterm labor, or significant disease in the surviving neonate. Exploration of the cellular and histological components of the placental barrier is in its infancy, and both how and where T. gondii breaches it are unknown. The human placenta presents two anatomical interfaces between maternal cells and fetal cells (trophoblasts): (i) the villous region where maternal blood bathes syncytialized trophoblasts for nutrient exchange and (ii) the maternal decidua, where mononuclear, extravillous trophoblasts anchor the villous region to the uterus. Using first-trimester human placental explants, we demonstrate that the latter site is significantly more vulnerable to infection, despite presenting a vastly smaller surface. This is consistent with past findings concerning two vertically transmitted viruses and one bacterium. We further explore whether three genetically distinct T. gondii types (I, II, and III) are capable of preferential placental infection and survival in this model. We find no difference in these strains' ability to infect placental explants; however, slightly slower growth is evident in type II (Prugniaud [Pru]) parasites relative to other cell types, although this did not quite achieve statistical significance.

  View details for DOI 10.1128/IAI.05899-11

  View details for Web of Science ID 000298402500042

  View details for PubMedID 22083708

• Identification of Tissue Cyst Wall Components by Transcriptome Analysis of In Vivo and In Vitro Toxoplasma gondii Bradyzoites EUKARYOTIC CELL Buchholz, K. R., Fritz, H. M., Chen, X., Durbin-Johnson, B., Rocke, D. M., Ferguson, D. J., Conrad, P. A., Boothroyd, J. C. 2011; 10 (12): 1637-1647

  Abstract

  The Toxoplasma gondii bradyzoite is essential to establish persistent infection, yet little is known about what factors this developmental form secretes to establish the cyst or interact with its host cell. To identify candidate bradyzoite-secreted effectors, the transcriptomes of in vitro tachyzoites 2 days postinfection, in vitro bradyzoites 4 days postinfection, and in vivo bradyzoites 21 days postinfection were interrogated by microarray, and the program SignalP was used to identify signal peptides indicating secretion. One hundred two putative bradyzoite-secreted effectors were identified by this approach. Two candidates, bradyzoite pseudokinase 1 and microneme adhesive repeat domain-containing protein 4, were chosen for further investigation and confirmed to be induced and secreted by bradyzoites in vitro and in vivo. Thus, we report the first analysis of the transcriptomes of in vitro and in vivo bradyzoites and identify two new protein components of the Toxoplasma tissue cyst wall.

  View details for DOI 10.1128/EC.05182-11

  View details for Web of Science ID 000298153500005

  View details for PubMedID 22021236

  View details for PubMedCentralID PMC3232729

• Toxoplasma gondii Induces B7-2 Expression through Activation of JNK Signal Transduction INFECTION AND IMMUNITY Morgado, P., Ong, Y., Boothroyd, J. C., Lodoen, M. B. 2011; 79 (11): 4401-4412

  Abstract

  Toxoplasma gondii is a globally distributed parasite pathogen that infects virtually all warm-blooded animals. A hallmark of immunity to acute infection is the production of gamma interferon (IFN-γ) and interleukin-12 (IL-12), followed by a protective T cell response that is critical for parasite control. Naïve T cell activation requires both T-cell receptor (TCR) stimulation and the engagement of costimulatory receptors. Because of their important function in activating T cells, the expression of costimulatory ligands is believed to be under tight control. The molecular mechanisms governing their induction during microbial stimulation, however, are not well understood. We found that all three strains of T. gondii (types I, II, and III) upregulated the expression of B7-2, but not B7-1, on the surface of mouse bone marrow-derived macrophages. Additionally, intraperitoneal infection of mice with green fluorescent protein (GFP)-expressing parasites resulted in enhanced B7-2 levels specifically on infected, GFP(+) CD11b(+) cells. B7-2 induction occurred at the transcript level, required active parasite invasion, and was not dependent on MyD88 or TRIF. Functional assays demonstrated that T. gondii-infected macrophages stimulated naïve T cell proliferation in a B7-2-dependent manner. Genome-wide transcriptional analysis comparing infected and uninfected macrophages revealed the activation of mitogen-activated protein kinase (MAPK) signaling in infected cells. Using specific inhibitors against MAPKs, we determined that parasite-induced B7-2 is dependent on Jun N-terminal protein kinase (JNK) but not extracellular signal-regulated kinase (ERK) or p38 signaling. We also observed that T. gondii-induced B7-2 expression on human peripheral blood monocytes is dependent on JNK signaling, indicating that a common mechanism of B7-2 regulation by T. gondii may exist in both humans and mice.

  View details for DOI 10.1128/IAI.05562-11

  View details for Web of Science ID 000296352400012

  View details for PubMedID 21911468

• The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries CELL HOST & MICROBE Treeck, M., Sanders, J. L., Elias, J. E., Boothroyd, J. C. 2011; 10 (4): 410-419

  Abstract

  Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.

  View details for DOI 10.1016/j.chom.2011.09.004

  View details for Web of Science ID 000296600700016

  View details for PubMedID 22018241

  View details for PubMedCentralID PMC3254672

• Strain-Dependent Host Transcriptional Responses to Toxoplasma Infection Are Largely Conserved in Mammalian and Avian Hosts PLOS ONE Ong, Y., Boyle, J. P., Boothroyd, J. C. 2011; 6 (10)

  Abstract

  Toxoplasma gondii has a remarkable ability to infect an enormous variety of mammalian and avian species. Given this, it is surprising that three strains (Types I/II/III) account for the majority of isolates from Europe/North America. The selective pressures that have driven the emergence of these particular strains, however, remain enigmatic. We hypothesized that strain selection might be partially driven by adaptation of strains for mammalian versus avian hosts. To test this, we examine in vitro, strain-dependent host responses in fibroblasts of a representative avian host, the chicken (Gallus gallus). Using gene expression profiling of infected chicken embryonic fibroblasts and pathway analysis to assess host response, we show here that chicken cells respond with distinct transcriptional profiles upon infection with Type II versus III strains that are reminiscent of profiles observed in mammalian cells. To identify the parasite drivers of these differences, chicken fibroblasts were infected with individual F1 progeny of a Type II x III cross and host gene expression was assessed for each by microarray. QTL mapping of transcriptional differences suggested, and deletion strains confirmed, that, as in mammalian cells, the polymorphic rhoptry kinase ROP16 is the major driver of strain-specific responses. We originally hypothesized that comparing avian versus mammalian host response might reveal an inversion in parasite strain-dependent phenotypes; specifically, for polymorphic effectors like ROP16, we hypothesized that the allele with most activity in mammalian cells might be less active in avian cells. Instead, we found that activity of ROP16 alleles appears to be conserved across host species; moreover, additional parasite loci that were previously mapped for strain-specific effects on mammalian response showed similar strain-specific effects in chicken cells. These results indicate that if different hosts select for different parasite genotypes, the selection operates downstream of the signaling occurring during the beginning of the host's immune response.

  View details for DOI 10.1371/journal.pone.0026369

  View details for Web of Science ID 000295978600051

  View details for PubMedID 22022607

  View details for PubMedCentralID PMC3192797

• Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication TRENDS IN PARASITOLOGY Tyler, J. S., Treeck, M., Boothroyd, J. C. 2011; 27 (9): 410-420

  Abstract

  Apicomplexan parasites exhibit an unusual mechanism of host cell penetration. A central player in this process is the protein apical membrane antigen 1 (AMA1). Although essential for invasion, the precise functional roles AMA1 plays have been unclear. Several recent studies have provided important functional insight into its role within the multiprotein complex that comprises the moving junction (MJ). Initially formed at the apical tip of the invading parasite, the MJ represents a ring-like region of contact between the surfaces of the invading parasite and the host cell as the invaginated host plasma membrane is forced inward by the penetrating parasite. This review discusses these and other recent insights into AMA1 with particular emphasis on studies conducted in Plasmodium and Toxoplasma.

  View details for DOI 10.1016/j.pt.2011.04.002

  View details for Web of Science ID 000295207500008

  View details for PubMedID 21659001

  View details for PubMedCentralID PMC3159806

• A Conserved Non-canonical Motif in the Pseudoactive Site of the ROP5 Pseudokinase Domain Mediates Its Effect on Toxoplasma Virulence JOURNAL OF BIOLOGICAL CHEMISTRY Reese, M. L., Boothroyd, J. C. 2011; 286 (33): 29366-29375

  Abstract

  The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATP-binding motifs for non-catalytic signaling through allostery.

  View details for DOI 10.1074/jbc.M111.253435

  View details for Web of Science ID 000293837000071

  View details for PubMedID 21708941

  View details for PubMedCentralID PMC3190742

• Binding of Plasmodium merozoite proteins RON2 and AMA1 triggers commitment to invasion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Srinivasan, P., Beatty, W. L., Diouf, A., Herrera, R., Ambroggio, X., Moch, J. K., Tyler, J. S., Narum, D. L., Pierce, S. K., Boothroyd, J. C., Haynes, J. D., Miller, L. H. 2011; 108 (32): 13275-13280

  Abstract

  The commitment of Plasmodium merozoites to invade red blood cells (RBCs) is marked by the formation of a junction between the merozoite and the RBC and the coordinated induction of the parasitophorous vacuole. Despite its importance, the molecular events underlying the parasite's commitment to invasion are not well understood. Here we show that the interaction of two parasite proteins, RON2 and AMA1, known to be critical for invasion, is essential to trigger junction formation. Using antibodies (Abs) that bind near the hydrophobic pocket of AMA1 and AMA1 mutated in the pocket, we identified RON2's binding site on AMA1. Abs specific for the AMA1 pocket blocked junction formation and the induction of the parasitophorous vacuole. We also identified the critical residues in the RON2 peptide (previously shown to bind AMA1) that are required for binding to the AMA1 pocket, namely, two conserved, disulfide-linked cysteines. The RON2 peptide blocked junction formation but, unlike the AMA1-specific Ab, did not block formation of the parasitophorous vacuole, indicating that formation of the junction and parasitophorous vacuole are molecularly distinct steps in the invasion process. Collectively, these results identify the binding of RON2 to the hydrophobic pocket of AMA1 as the step that commits Plasmodium merozoites to RBC invasion and point to RON2 as a potential vaccine candidate.

  View details for DOI 10.1073/pnas.1110303108

  View details for Web of Science ID 000293691400063

  View details for PubMedID 21788485

• Evidence for Host Cells as the Major Contributor of Lipids in the Intravacuolar Network of Toxoplasma-Infected Cells EUKARYOTIC CELL Caffaro, C. E., Boothroyd, J. C. 2011; 10 (8): 1095-1099

  Abstract

  The intracellular parasite Toxoplasma gondii develops inside a parasitophorous vacuole (PV) that derives from the host cell plasma membrane during invasion. Previous electron micrograph images have shown that the membrane of this vacuole undergoes an extraordinary remodeling with an extensive network of thin tubules and vesicles, the intravacuolar network (IVN), which fills the lumen of the PV. While dense granule proteins, secreted during and after invasion, are the main factors for the organization and tubulation of the network, little is known about the source of lipids used for this remodeling. By selectively labeling host cell or parasite membranes, we uncovered evidence that strongly supports the host cell as the primary, if not exclusive, source of lipids for parasite IVN remodeling. Fluorescence recovery after photobleaching (FRAP) microscopy experiments revealed that lipids are surprisingly dynamic within the parasitophorous vacuole and are continuously exchanged or replenished by the host cell. The results presented here suggest a new model for development of the parasitophorous vacuole whereby the host provides a continuous stream of lipids to support the growth and maturation of the PVM and IVN.

  View details for DOI 10.1128/EC.00002-11

  View details for Web of Science ID 000293383000010

  View details for PubMedID 21685319

  View details for PubMedCentralID PMC3165450

• Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hall, C. I., Reese, M. L., Weerapana, E., Child, M. A., Bowyer, P. W., Albrow, V. E., Haraldsen, J. D., Phillips, M. R., Sandoval, E. D., Ward, G. E., Cravatt, B. F., Boothroyd, J. C., Bogyo, M. 2011; 108 (26): 10568-10573

  Abstract

  Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

  View details for DOI 10.1073/pnas.1105622108

  View details for Web of Science ID 000292251000042

  View details for PubMedID 21670272

  View details for PubMedCentralID PMC3127939

• Toxoplasma Polymorphic Effectors Determine Macrophage Polarization and Intestinal Inflammation CELL HOST & MICROBE Jensen, K. D., Wang, Y., Wojno, E. D., Shastri, A. J., Hu, K., Cornel, L., Boedec, E., Ong, Y., Chien, Y., Hunter, C. A., Boothroyd, J. C., Saeij, J. P. 2011; 9 (6): 472-483

  Abstract

  European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate macrophage activation influence the ability of Toxoplasma to establish a chronic infection.

  View details for DOI 10.1016/j.chom.2011.04.015

  View details for Web of Science ID 000293157200006

  View details for PubMedID 21669396

  View details for PubMedCentralID PMC3131154

• Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Reese, M. L., Zeiner, G. M., Saeij, J. P., Boothroyd, J. C., Boyle, J. P. 2011; 108 (23): 9625-9630

  Abstract

  Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4-10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma's successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

  View details for DOI 10.1073/pnas.1015980108

  View details for Web of Science ID 000291341400062

  View details for PubMedID 21436047

  View details for PubMedCentralID PMC3111280

• Chemistry and Biology of Macrolide Antiparasitic Agents JOURNAL OF MEDICINAL CHEMISTRY Lee, Y., Choi, J. Y., Fu, H., Harvey, C., Ravindran, S., Roush, W. R., Boothroyd, J. C., Khosla, C. 2011; 54 (8): 2792-2804

  Abstract

  Macrolide antibacterial agents inhibit parasite proliferation by targeting the apicoplast ribosome. Motivated by the long-term goal of identifying antiparasitic macrolides that lack antibacterial activity, we have systematically analyzed the structure-activity relationships among erythromycin analogues and have also investigated the mechanism of action of selected compounds. Two lead compounds, N-benzylazithromycin (11) and N-phenylpropylazithromycin (30), were identified with significantly higher antiparasitic activity and lower antibacterial activity than erythromycin or azithromycin. Molecular modeling based on the cocrystal structure of azithromycin bound to the bacterial ribosome suggested that a substituent at the N-9 position of desmethylazithromycin could improve selectivity because of species-specific interactions with the ribosomal L22 protein. Like other macrolides, these lead compounds display a strong "delayed death phenotype"; however, their early effects on T. gondii replication are more pronounced.

  View details for DOI 10.1021/jm101593u

  View details for Web of Science ID 000289697800018

  View details for PubMedID 21428405

  View details for PubMedCentralID PMC3085955

• The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex PLOS PATHOGENS Tyler, J. S., Boothroyd, J. C. 2011; 7 (2)

  Abstract

  Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.

  View details for DOI 10.1371/journal.ppat.1001282

  View details for Web of Science ID 000287698200020

  View details for PubMedID 21347354

  View details for PubMedCentralID PMC3037364

• Association of host mitochondria with the parasitophorous vacuole during Toxoplasma infection is not dependent on rhoptry proteins ROP2/8 INTERNATIONAL JOURNAL FOR PARASITOLOGY Pernas, L., Boothroyd, J. C. 2010; 40 (12): 1367-1371

  Abstract

  Previous work has proposed rhoptry protein 2 (ROP2) as the physical link that tethers host mitochondria to the parasitophorous vacuole membrane (PVM) surrounding the intracellular parasite, Toxoplasma gondii. A recent analysis of the ROP2 structure, however, raised questions about this model. To determine whether ROP2 is necessary, we created a parasite line that lacks the entire ROP2 locus consisting of the three closely related genes, ROP2a, ROP2b and ROP8. We show that this knockout mutant retains the ability to recruit host mitochondria in a manner that is indistinguishable from the parental strain, re-opening the question of which molecules mediate this association.

  View details for DOI 10.1016/j.ijpara.2010.07.002

  View details for Web of Science ID 000282867200002

  View details for PubMedID 20637758

  View details for PubMedCentralID PMC2939271

• Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6 JOURNAL OF BIOLOGICAL CHEMISTRY Ong, Y., Reese, M. L., Boothroyd, J. C. 2010; 285 (37): 28731-28740

  Abstract

  The obligate intracellular parasite, Toxoplasma gondii, modulates host immunity in a variety of highly specific ways. Previous work revealed a polymorphic, injected parasite factor, ROP16, to be a key virulence determinant and regulator of host cell transcription. These properties were shown to be partially mediated by dysregulation of the host transcription factors STAT3 and STAT6, but the molecular mechanisms underlying this phenotype were unclear. Here, we use a Type I Toxoplasma strain deficient in ROP16 to show that ROP16 induces not only sustained activation but also an extremely rapid (within 1 min) initial activation of STAT6. Using recombinant wild-type and kinase-deficient ROP16, we demonstrate in vitro that ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation, Tyr(641). Furthermore, ROP16 co-immunoprecipitates with STAT6 from infected cells. Taken together, these data strongly suggest that STAT6 is a direct substrate for ROP16 in vivo.

  View details for DOI 10.1074/jbc.M110.112359

  View details for Web of Science ID 000281594000039

  View details for PubMedID 20624917

  View details for PubMedCentralID PMC2937901

• Coordinated loading of IRG resistance GTPases on to the Toxoplasma gondii parasitophorous vacuole CELLULAR MICROBIOLOGY Khaminets, A., Hunn, J. P., Koenen-Waisman, S., Zhao, Y. O., Preukschat, D., Coers, J., Boyle, J. P., Ong, Y., Boothroyd, J. C., Reichmann, G., Howard, J. C. 2010; 12 (7): 939-961

  Abstract

  The immunity-related GTPases (IRGs) constitute an interferon-induced intracellular resistance mechanism in mice against Toxoplasma gondii. IRG proteins accumulate on the parasitophorous vacuole membrane (PVM), leading to its disruption and to death of the parasite. How IRGs target the PVM is unknown. We show that accumulation of IRGs on the PVM begins minutes after parasite invasion and increases for about 1 h. Targeting occurs independently of several signalling pathways and the microtubule network, suggesting that IRG transport is diffusion-driven. The intensity of IRG accumulation on the PVM, however, is reduced in absence of the autophagy regulator, Atg5. In wild-type cells IRG proteins accumulate cooperatively on PVMs in a definite order reflecting a temporal hierarchy, with Irgb6 and Irgb10 apparently acting as pioneers. Loading of IRG proteins onto the vacuoles of virulent Toxoplasma strains is attenuated and the two pioneer IRGs are the most affected. The polymorphic rhoptry kinases, ROP16, ROP18 and the catalytically inactive proteins, ROP5A-D, are not individually responsible for this effect. Thus IRG proteins protect mice against avirulent strains of Toxoplasma but fail against virulent strains. The complex cooperative behaviour of IRG proteins in resisting Toxoplasma may hint at undiscovered complexity also in virulence mechanisms.

  View details for DOI 10.1111/j.1462-5822.2010.01443.x

  View details for Web of Science ID 000278518900008

  View details for PubMedID 20109161

  View details for PubMedCentralID PMC2901525

• Use of two novel approaches to discriminate between closely related host microRNAs that are manipulated by Toxoplasma gondii during infection RNA-A PUBLICATION OF THE RNA SOCIETY Zeiner, G. M., Boothroyd, J. C. 2010; 16 (6): 1268-1274

  Abstract

  MicroRNAs (miRNAs) are a class of small, endogenously encoded regulatory RNAs that function to post-transcriptionally regulate gene expression in a wide variety of eukaryotes. Within organisms, some mature miRNAs, such as paralogous miRNAs, have nearly identical nucleotide sequences, which makes them virtually indistinguishable from one another by conventional hybridization-based approaches. Here we describe two inexpensive, sensitive methods for rapidly discriminating between paralogous miRNAs or other closely related miRNAs and for quantifying their abundance. The first approach is a sequential ribonuclease-protection and primer-extension assay; the second approach is a primer-extension assay that employs short oligonucleotide probes to exacerbate the instability of mismatched probe:miRNA hybrids. Both approaches are rapid and can be easily performed in their entirety using common laboratory equipment. As a proof of concept, we have used these methods to determine the exact identities of the human miR-17 family members that are increased by infection with the intracellular parasite Toxoplasma gondii. These methods can be used to rapidly and inexpensively discriminate between any closely related miRNAs in any organism.

  View details for DOI 10.1261/rna.2069310

  View details for Web of Science ID 000278125600019

  View details for PubMedID 20423977

  View details for PubMedCentralID PMC2874178

• Toxoplasma secreting Cre recombinase for analysis of host-parasite interactions NATURE METHODS Koshy, A. A., Fouts, A. E., Lodoen, M. B., Alkan, O., Blau, H. M., Boothroyd, J. C. 2010; 7 (4): 307-309

  Abstract

  We describe a Toxoplasma gondii strain that will permit the use of site-specific recombination to study the host-parasite interactions of this organism. This Toxoplasma strain efficiently injects a Cre fusion protein into host cells. In a Cre-reporter cell line, a single parasite invasion induced Cre-mediated recombination in 95% of infected host cells. By infecting Cre-reporter mice with these parasites, we also monitored host-cell infection in vivo.

  View details for DOI 10.1038/NMETH.1438

  View details for Web of Science ID 000276150600022

  View details for PubMedID 20208532

  View details for PubMedCentralID PMC2850821

• Toxoplasma gondii Infection Specifically Increases the Levels of Key Host MicroRNAs PLOS ONE Zeiner, G. M., Norman, K. L., Thomson, J. M., Hammond, S. M., Boothroyd, J. C. 2010; 5 (1)

  Abstract

  The apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection.Using microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection.Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

  View details for DOI 10.1371/journal.pone.0008742

  View details for Web of Science ID 000273714900022

  View details for PubMedID 20090903

  View details for PubMedCentralID PMC2806928

• A highly sensitive FRET-based approach reveals secretion of the actin-binding protein toxofilin during Toxoplasma gondii infection CELLULAR MICROBIOLOGY Lodoen, M. B., Gerke, C., Boothroyd, J. C. 2010; 12 (1): 55-66

  Abstract

  We have utilized a highly sensitive approach based on fluorescence resonance energy transfer (FRET) and beta-lactamase (BLA), which we adapted for the detection of Toxoplasma gondii secreted proteins. This assay revealed that the actin-binding protein toxofilin appears to be secreted into host cells during invasion. To determine the function of toxofilin during infection, we engineered a type I (RH strain) parasite with a targeted deletion of the toxofilin gene and compared the phenotypes of control and toxofilin knockout (Deltatxf) parasites in several in vitro assays, including invasion, growth, gliding motility, and egress of the Deltatxf parasites, as well as F-actin staining, phagocytosis and migration of cells infected with Deltatxf parasites or wild-type controls. Despite its apparent secretion into host cells and its ability to bind to and modulate host actin, we observed that toxofilin does not appear to play a role in these processes, under the conditions we examined, and we report these findings here.

  View details for DOI 10.1111/j.1462-5822.2009.01378.x

  View details for Web of Science ID 000273162200006

  View details for PubMedID 19732057

  View details for PubMedCentralID PMC3411547

• 4-Bromophenacyl Bromide Specifically Inhibits Rhoptry Secretion during Toxoplasma Invasion PLOS ONE Ravindran, S., Lodoen, M. B., Verhelst, S. H., Bogyo, M., Boothroyd, J. C. 2009; 4 (12)

  Abstract

  Toxoplasma gondii is a eukaryotic parasite of the phylum Apicomplexa that is able to infect a wide variety of host cells. During its active invasion process it secretes proteins from discrete secretory organelles: the micronemes, rhoptries and dense granules. Although a number of rhoptry proteins have been shown to be involved in important interactions with the host cell, very little is known about the mechanism of secretion of any Toxoplasma protein into the host cell. We used a chemical inhibitor of phospholipase A2s, 4-bromophenacyl bromide (4-BPB), to look at the role of such lipases in the secretion of Toxoplasma proteins. We found that 4-BPB was a potent inhibitor of rhoptry secretion in Toxoplasma invasion. This drug specifically blocked rhoptry secretion but not microneme secretion, thus effectively showing that the two processes can be de-coupled. It affected parasite motility and invasion, but not attachment or egress. Using propargyl- or azido-derivatives of the drug (so-called click chemistry derivatives) and a series of 4-BPB-resistant mutants, we found that the drug has a very large number of target proteins in the parasite that are involved in at least two key steps: invasion and intracellular growth. This potent compound, the modified "click-chemistry" forms of it, and the resistant mutants should serve as useful tools to further study the processes of Toxoplasma early invasion, in general, and rhoptry secretion, in particular.

  View details for DOI 10.1371/journal.pone.0008143

  View details for Web of Science ID 000272828800031

  View details for PubMedID 19956582

  View details for PubMedCentralID PMC2780294

• A Helical Membrane-Binding Domain Targets the Toxoplasma ROP2 Family to the Parasitophorous Vacuole TRAFFIC Reese, M. L., Boothroyd, J. C. 2009; 10 (10): 1458-1470

  Abstract

  During invasion, the obligate intracellular pathogen, Toxoplasma gondii, secretes into its host cell a variety of effector molecules, several of which have been implicated in strain-specific variation in disease. The largest family of these effectors, defined by the canonical member ROP2, quickly associates with the nascent parasitophorous vacuole membrane (PVM) after secretion. Here we demonstrate that the NH(2)-terminal domain of the ROP2 family contains a series of amphipathic helices that are necessary and sufficient for membrane association. While each of the amphipathic helices is individually competent to bind cellular membranes, together they act to bind the PVM preferentially, possibly through sensing its strong negative curvature. This previously uncharacterized helical domain is an evolutionarily robust and energetically efficient design for membrane association.

  View details for DOI 10.1111/j.1600-0854.2009.00958.x

  View details for Web of Science ID 000269732500007

  View details for PubMedID 19682324

  View details for PubMedCentralID PMC2746882

• Toxoplasma gondii: 25 years and 25 major advances for the field INTERNATIONAL JOURNAL FOR PARASITOLOGY Boothroyd, J. C. 2009; 39 (8): 935-946

  Abstract

  This article is an attempt to identify the most significant highlights of Toxoplasma research over the last 25 years. It has been a period of enormous progress and the top 25 most significant advances, in the view of this author, are described. These range from the bench to the bedside and represent a tremendous body of work from countless investigators. And, having laid out so much that has been discovered, it is impossible not to also reflect on the challenges that lie ahead. These, too, are briefly discussed. Finally, while every effort has been made to view the field as a whole, the molecular biology background of the author almost certainly will have skewed the relative importance attached to past and future advances. Despite this, it is hoped that the reader will agree with, or at least not disagree too strongly with, most of the choices presented here.

  View details for DOI 10.1016/j.ijpara.2009.02.003

  View details for PubMedID 19630140

• A Pseudouridine Synthase Homologue Is Critical to Cellular Differentiation in Toxoplasma gondii EUKARYOTIC CELL Anderson, M. Z., Brewer, J., Singh, U., Boothroyd, J. C. 2009; 8 (3): 398-409

  Abstract

  Toxoplasma gondii is a haploid protozoan parasite infecting about one in seven people in the United States. Key to the worldwide prevalence of T. gondii is its ability to establish a lifelong, chronic infection by evading the immune system, and central to this is the developmental switch between the two asexual forms, tachyzoites and bradyzoites. A library of mutants defective in tachyzoite-to-bradyzoite differentiation (Tbd(-)) was created through insertional mutagenesis. This library contains mutants that, compared to the wild type, are between 20% and 74% as efficient at stage conversion. Two mutants, TBD5 and TBD8, with disruptions in a gene encoding a putative pseudouridine synthase, PUS1, were identified. The disruption in TBD8 is in the 5' end of the PUS1 gene and appears to produce a null allele with a 50% defect in differentiation. This is about the same switch efficiency as obtained with an engineered pus1 deletion mutant (Deltapus1). The insertion in TBD5 is within the PUS1 coding region, and this appears to result in a more extreme phenotype of only approximately 10% switch efficiency. Complementation of TBD8 with the genomic PUS1 allele restored wild-type differentiation efficiency. Infection of mice with pus1 mutant strains results in increased mortality during the acute phase and higher cyst burdens during the chronic infection, demonstrating an aberrant differentiation phenotype in vivo due to PUS1 disruption. Our results suggest a surprising and important role for RNA modification in this biological process.

  View details for DOI 10.1128/EC.00329-08

  View details for Web of Science ID 000263935200014

  View details for PubMedID 19124578

  View details for PubMedCentralID PMC2653242

• Expansion of host range as a driving force in the evolution of Toxoplasma Meeting on Toxoplasma Centennial Congress from Discovery to Public Health Management Boothroyd, J. C. FUNDACO OSWALDO CRUZ. 2009: 179–84

  Abstract

  The apicomplexan parasite Toxoplasma gondii is unusual in being able to infect almost any cell from almost any warm-blooded animal it encounters. This extraordinary host-range contrasts with its far more particular cousins such as the various species of the malaria parasite Plasmodium where each species of parasite has a single genus or even species of host that it can infect. Genetic and genomic studies have revealed a key role for a number of gene families in how Toxoplasma invades a host cell, modulates gene expression of that cell and successfully evades the resulting immune response. In this review, I will explore the hypothesis that a combination of sexual recombination and expansion of host range may be the major driving forces in the evolution of some of these gene families and the specific genes they encompass. These ideas stem from results and thoughts published by several labs in the last few years but especially recent papers on the role of different forms of rhoptry proteins in the relative virulence of F1 Toxoplasma progeny in a particular host species (mice).

  View details for Web of Science ID 000267051500009

  View details for PubMedID 19430641

• Rapid Membrane Disruption by a Perforin-Like Protein Facilitates Parasite Exit from Host Cells SCIENCE Kafsack, B. F., Pena, J. D., Coppens, I., Ravindran, S., Boothroyd, J. C., Carruthers, V. B. 2009; 323 (5913): 530-533

  Abstract

  Perforin-like proteins are expressed by many bacterial and protozoan pathogens, yet little is known about their function or mode of action. Here, we describe Toxoplasma perforin-like protein 1 (TgPLP1), a secreted perforin-like protein of the intracellular protozoan pathogen Toxoplasma gondii that displays structural features necessary for pore formation. After intracellular growth, TgPLP1-deficient parasites failed to exit normally, resulting in entrapment within host cells. We show that this defect is due to an inability to rapidly permeabilize the parasitophorous vacuole membrane and host plasma membrane during exit. TgPLP1 ablation had little effect on growth in culture but resulted in a reduction greater than five orders of magnitude of acute virulence in mice. Perforin-like proteins from other intracellular pathogens may play a similar role in microbial egress and virulence.

  View details for DOI 10.1126/science.1165740

  View details for Web of Science ID 000262587900050

  View details for PubMedID 19095897

  View details for PubMedCentralID PMC2662845

• The Toxoplasma gondii Dense Granule Protein GRA7 Is Phosphorylated upon Invasion and Forms an Unexpected Association with the Rhoptry Proteins ROP2 and ROP4 INFECTION AND IMMUNITY Dunn, J. D., Ravindran, S., Kim, S., Boothroyd, J. C. 2008; 76 (12): 5853-5861

  Abstract

  The obligate intracellular parasite Toxoplasma gondii infects warm-blooded animals throughout the world and is an opportunistic pathogen of humans. As it invades a host cell, Toxoplasma forms a novel organelle, the parasitophorous vacuole, in which it resides during its intracellular development. The parasite modifies the parasitophorous vacuole and its host cell with numerous proteins delivered from rhoptries and dense granules, which are secretory organelles unique to the phylum Apicomplexa. For the majority of these proteins, little is known other than their localization. Here we show that the dense granule protein GRA7 is phosphorylated but only in the presence of host cells. Within 10 min of invasion, GRA7 is present in strand-like structures in the host cytosol that contain rhoptry proteins. GRA7 strands also contain GRA1 and GRA3. Independently of its phosphorylation state, GRA7 associates with the rhoptry proteins ROP2 and ROP4 in infected host cells. This is the first report of interactions between proteins secreted from rhoptries and dense granules.

  View details for DOI 10.1128/IAI.01667-07

  View details for Web of Science ID 000261003900045

  View details for PubMedID 18809661

  View details for PubMedCentralID PMC2583583

• Expression quantitative trait locus mapping of Toxoplasma genes reveals multiple mechanisms for strain-specific differences in gene expression EUKARYOTIC CELL Boyle, J. P., Saeij, J. P., Harada, S. Y., Ajioka, J. W., Boothroyd, J. C. 2008; 7 (8): 1403-1414

  Abstract

  Toxoplasma gondii is an intracellular parasite with a significant impact on human health, especially in cases where individuals are immunocompromised (e.g., due to human immunodeficiency virus/AIDS). In Europe and North America, only a few clonal genotypes appear to be responsible for the vast majority of Toxoplasma infections, and these clonotypes have been intensely studied to identify strain-specific phenotypes that may play a role in the manifestation of more-severe disease. To identify and genetically map strain-specific differences in gene expression, we have carried out expression quantitative trait locus analysis on Toxoplasma gene expression phenotypes by using spotted cDNA microarrays. This led to the identification of 16 Toxoplasma genes that had significant and mappable strain-specific variation in hybridization intensity. While the analysis should identify both cis- and trans-mapping hybridization profiles, we identified only loci with strain-specific hybridization differences that are most likely due to differences in the locus itself (i.e., cis mapping). Interestingly, a larger number of these cis-mapping genes than would be expected by chance encode either confirmed or predicted secreted proteins, many of which are known to localize to the specialized secretory organelles characteristic of members of the phylum Apicomplexa. For six of the cis-mapping loci, we determined if the strain-specific hybridization differences were due to true transcriptional differences or rather to strain-specific differences in hybridization efficiency because of extreme polymorphism and/or deletion, and we found examples of both scenarios.

  View details for DOI 10.1128/EC.00073-08

  View details for Web of Science ID 000258666500017

  View details for PubMedID 18552283

  View details for PubMedCentralID PMC2519772

• A cluster of four surface antigen genes specifically expressed in bradyzoites, SAG2CDXY, plays an important role in Toxoplasma gondii persistence INFECTION AND IMMUNITY Saeij, J. P., Arrizabalaga, G., Boothroyd, J. C. 2008; 76 (6): 2402-2410

  Abstract

  Toxoplasma gondii is one of the most successful protozoan parasites of warm-blooded animals. Stage-specific expression of its surface molecules is thought to be key to its ability to establish chronic infection in immunocompetent animals. The rapidly dividing tachyzoite stage displays a different subset of family of surface antigen 1 (SAG1)-related sequences (SRSs) from that displayed by the encysted bradyzoite stage. It is possible that this switch is necessary to protect the bradyzoites against an immune response raised against the tachyzoite stage. Alternatively, it might be that bradyzoite SRSs evolved to facilitate invasion of different cell types, such as those found in the brain, where cysts develop, or the small intestine, where bradyzoites must enter after oral infection. Here we studied the function of a cluster of four tandem genes, encoding bradyzoite SRSs called SAG2C, -D, -X, and -Y. Using bioluminescence imaging of mice infected with parasites expressing firefly luciferase (FLUC) driven by the SAG2D promoter, we show stage conversion for the first time in living animals. A truncated version of the SAG2D promoter (SAG2Dmin) gave efficient expression of FLUC in both tachyzoites and bradyzoites, indicating that the bradyzoite specificity of the complete SAG2D promoter is likely due to an element(s) that normally suppresses expression in tachyzoites. Comparing mice infected with the wild type or a mutant where the SAG2CDXY cluster of genes has been deleted (DeltaSAG2CDXY), we demonstrate that whereas DeltaSAG2CDXY parasites are less capable of maintaining a chronic infection in the brain, they do not show a defect in oral infectivity.

  View details for DOI 10.1128/IAI.01494-07

  View details for Web of Science ID 000256128900015

  View details for PubMedID 18347037

  View details for PubMedCentralID PMC2423105

• Secretion of proteins into host cells by Apicomplexan parasites TRAFFIC Ravindran, S., Boothroyd, J. C. 2008; 9 (5): 647-656

  Abstract

  The phylum Apicomplexa consists of a diverse group of obligate, intracellular parasites. The distinct evolutionary pressures on these protozoans as they have adapted to their respective niches have resulted in a variety of methods that they use to interact with and modify their hosts. One of these is the secretion and trafficking of parasite proteins into the host cell. We review this process for Theileria, Toxoplasma and Plasmodium. We also present what is known about the mechanisms by which parasite proteins are exported into the host cell, as well as information on their known and putative functions once they have reached their final destination.

  View details for DOI 10.1111/j.1600-0854.2008.00723.x

  View details for Web of Science ID 000254793000004

  View details for PubMedID 18266908

• Kiss and spit: the dual roles of Toxoplasma rhoptries NATURE REVIEWS MICROBIOLOGY Boothroyd, J. C., Dubremetz, J. 2008; 6 (1): 79-88

  Abstract

  Toxoplasma gondii is a single-celled, eukaryotic parasite that can only reproduce inside a host cell. Upon entry, this Apicomplexan parasite co-opts host functions for its own purposes. An unusual set of apical organelles, named rhoptries, contain some of the machinery that is used by T. gondii both for invasion and to commandeer host functions. Of particular interest are a group of injected protein kinases that are among the most variable of all the T. gondii proteins. At least one of these kinases has a major effect on host-gene expression, including the modulation of key regulators of the immune response. Here, we discuss these recent findings and use them to propose a model in which an expansion of host range is a major force that drives rhoptry-protein evolution.

  View details for DOI 10.1038/nrmicro1800

  View details for Web of Science ID 000252065900016

  View details for PubMedID 18059289

• RNA analysis by biosynthetic tagging using 4-thiouracil and uracil phosphoribosyltransferase. Methods in molecular biology (Clifton, N.J.) Zeiner, G. M., Cleary, M. D., Fouts, A. E., Meiring, C. D., Mocarski, E. S., Boothroyd, J. C. 2008; 419: 135-146

  Abstract

  RNA analysis by biosynthetic tagging (RABT) enables sensitive and specific queries of (a) how gene expression is regulated on a genome-wide scale and (b) transcriptional profiling of a single cell or tissue type in vivo. RABT can be achieved by exploiting unique properties of Toxoplasma gondii uracil phosphoribosyltransferase (TgUPRT), a pyrimidine salvage enzyme that couples ribose-5-phosphate to the N1 nitrogen of uracil to yield uridine monophosphate (UMP). When 4-thiouracil is provided as a TgUPRT substrate, the resultant product is 4-thiouridine monophosphate which can, ultimately, be incorporated into RNA. Thio-substituted nucleotides are not a natural component of nucleic acids and are readily tagged, detected, and purified with commercially available reagents. Thus, one can do pulse/chase experiments to measure synthesis and decay rates and/or use cell-specific expression of the TgUPRT to tag only RNA synthesized in a given cell type. This chapter updates the original RABT protocol (1) and addresses methodological details associated with RABT that were beyond the scope or space allotment of the initial report.

  View details for DOI 10.1007/978-1-59745-033-1_9

  View details for PubMedID 18369980

• Toxoplasma gondii: Inconsistent dissemination patterns following oral infection in mice EXPERIMENTAL PARASITOLOGY Boyle, J. P., Saeij, J. P., Boothroyd, J. C. 2007; 116 (3): 302-305

  Abstract

  Since Toxoplasma gondii is transmitted in the wild through the ingestion of infective cysts, oral infection is a preferred model for studying the natural mode of parasite dissemination and pathogenesis. Using luciferase-expressing strains of T. gondii and in vivo imaging, we observed different patterns of disease progression in mice depending of the method of oral infection. Oral gavage of infective cysts (e.g., bradyzoites) resulted in an inconsistent pattern of parasite dissemination; in the majority (20/29) of infected mice, luciferase-derived signal (indicating high numbers of Toxoplasma tachyzoites) was first observed in the right chest area. At later time points this signal spread to other parts of the mouse, including the abdominal area. In the remaining mice (9/29), parasites were first observed replicating in the abdominal area, as might be expected. In contrast, when mice were infected naturally (either via ingestion of whole brains from previously infected mice or brain cyst homogenate-soaked bread), parasites were first observed replicating in the abdominal area in all mice examined (10/10). Based on the inconsistency of infections initiated with oral gavage, it is recommended that natural feeding be used to infect mice when a consistent oral infection is desired.

  View details for DOI 10.1016/j.exppara.2007.01.010

  View details for Web of Science ID 000247662500015

  View details for PubMedID 17335814

• The BSR4 protein is up-regulated in Toxoplasma gondii bradyzoites, however the dominant surface antigen recognised by the P36 monoclonal antibody is SRS9 INTERNATIONAL JOURNAL FOR PARASITOLOGY Van, T. T., Kim, S., Camps, M., Boothroyd, J. C., Knoll, L. J. 2007; 37 (8-9): 877-885

  Abstract

  The protozoan parasite, Toxoplasma gondii, interconverts between fast-growing tachyzoites and slow-growing bradyzoites within intermediate hosts. The surface of T. gondii is covered by the SAG1-related sequence (SRS) superfamily of glycosyl phosphatidyl inositol-anchored proteins, many of which are stage-specific. Previous transient transfection of BSR4, a member of the SRS superfamily, showed reactivity with the bradyzoite-specific P36 mAb by immunofluorescene assay. BSR4 mRNA levels were equally abundant in tachyzoites and bradyzoites, suggesting post-transcriptional regulation of the protein. In this study, we show that BSR4 protein is present in both tachyzoites and bradyzoites, but up-regulated in bradyzoites. However, stable expression of BSR4 in two BSR4-negative T. gondii strains shows minimal reactivity to the P36 mAb by Western immunoblotting, even though the BSR4 protein is abundant. We discovered that the SRS9 protein, a bradyzoite-specific member of the SRS superfamily and encoded immediately downstream of BSR4, was also ablated in the BSR4-negative strains, suggesting that SRS9 is the surface antigen recognised by the P36 mAb. Stable expression of SRS9 in the BSR4 mutant strains shows robust reactivity to the P36 mAb. Immunoprecipitation experiments confirm that the P36 mAb interacts with the SRS9 protein. These data indicate that while the BSR4 protein is up-regulated in bradyzoites, the dominant antigen that the P36 mAb recognises is SRS9.

  View details for DOI 10.1016/j.ijpara.2007.02.001

  View details for Web of Science ID 000248068800007

  View details for PubMedID 17368655

• Toxoplasma gondii dysregulates IFN-gamma-inducible gene expression in human fibroblasts: Insights from a genome-wide transcriptional profiling JOURNAL OF IMMUNOLOGY Kim, S., Fouts, A. E., Boothroyd, J. C. 2007; 178 (8): 5154-5165

  Abstract

  Toxoplasma gondii is an obligate intracellular parasite that persists for the life of a mammalian host. The parasite's ability to block the potent IFN-gamma response may be one of the key mechanisms that allow Toxoplasma to persist. Using a genome-wide microarray analysis, we show here a complete dysregulation of IFN-gamma-inducible gene expression in human fibroblasts infected with Toxoplasma. Notably, 46 of the 127 IFN-gamma-responsive genes were induced and 19 were suppressed in infected cells before they were exposed to IFN-gamma, indicating that other stimuli produced during infection may also regulate these genes. Following IFN-gamma treatment, none of the 127 IFN-gamma-responsive genes could be significantly induced in infected cells. Immunofluorescence assays showed at single-cell levels that infected cells, regardless of which Toxoplasma strain was used, could not be activated by IFN-gamma to up-regulate the expression of IFN regulatory factor 1, a transcription factor that is under the direct control of STAT1, whereas uninfected cells in the same culture expressed IFN regulatory factor 1 normally in response to IFN-gamma. STAT1 trafficked to the nucleus normally and indistinguishably in all uninfected and infected cells treated with IFN-gamma, indicating that the inhibitory effects of Toxoplasma infection likely occur via blocking STAT1 transcriptional activity in the nucleus. In contrast, a closely related apicomplexan, Neospora caninum, was unable to inhibit IFN-gamma-induced gene expression. A differential ability to interfere with the IFN-gamma response may, in part, account for the differences in the pathogenesis seen among Toxoplasma and Neospora parasite strains.

  View details for Web of Science ID 000245605300058

  View details for PubMedID 17404298

• Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vyas, A., Kim, S., Giacomini, N., Boothroyd, J. C., Sapolsky, R. M. 2007; 104 (15): 6442-6447

  Abstract

  The protozoan parasite Toxoplasma gondii blocks the innate aversion of rats for cat urine, instead producing an attraction to the pheromone; this may increase the likelihood of a cat predating a rat. This is thought to reflect adaptive, behavioral manipulation by Toxoplasma in that the parasite, although capable of infecting rats, reproduces sexually only in the gut of the cat. The "behavioral manipulation" hypothesis postulates that a parasite will specifically manipulate host behaviors essential for enhancing its own transmission. However, the neural circuits implicated in innate fear, anxiety, and learned fear all overlap considerably, raising the possibility that Toxoplasma may disrupt all of these nonspecifically. We investigated these conflicting predictions. In mice and rats, latent Toxoplasma infection converted the aversion to feline odors into attraction. Such loss of fear is remarkably specific, because infection did not diminish learned fear, anxiety-like behavior, olfaction, or nonaversive learning. These effects are associated with a tendency for parasite cysts to be more abundant in amygdalar structures than those found in other regions of the brain. By closely examining other types of behavioral patterns that were predicted to be altered we show that the behavioral effect of chronic Toxoplasma infection is highly specific. Overall, this study provides a strong argument in support of the behavioral manipulation hypothesis. Proximate mechanisms of such behavioral manipulations remain unknown, although a subtle tropism on part of the parasite remains a potent possibility.

  View details for DOI 10.1073/pnas.0608310104

  View details for Web of Science ID 000245737500062

  View details for PubMedID 17404235

  View details for PubMedCentralID PMC1851063

• Bradyzoite-specific surface antigen SRS9 plays a role in maintaining Toxoplasma gondii persistence in the brain and in host control of parasite replication in the intestine INFECTION AND IMMUNITY Kim, S., Karasov, A., Boothroyd, J. C. 2007; 75 (4): 1626-1634

  Abstract

  Toxoplasma gondii is a ubiquitous parasite that persists for the life of a healthy mammalian host. A latent, chronic infection can reactivate upon immunosuppression and cause life-threatening diseases, such as encephalitis. A key to the pathogenesis is the parasite's interconversion between the tachyzoite (in acute infection) and bradyzoite (in chronic infection) stages. This developmental switch is marked by differential expression of numerous, closely related surface proteins belonging to the SRS (SAG1-related sequence) superfamily. To probe the functions of bradyzoite-specific SRSs, we created a bioluminescent strain lacking the expression of SRS9, one of the most abundant SRSs of the bradyzoite stage. Imaging of mice intraperitoneally infected with tachyzoites revealed that during an acute infection, wild-type and Deltasrs9 strains replicated at similar rates, disseminated systemically following similar kinetics, and initially yielded similar brain cyst numbers. However, during a chronic infection, Deltasrs9 cyst loads substantially decreased compared to those of the wild type, suggesting that SRS9 plays a role in maintaining parasite persistence in the brain. In oral infection with bradyzoite cysts, the Deltasrs9 strain showed oral infectivity and dissemination patterns indistinguishable from those of the wild type. When chronically infected mice were treated with the immunosuppressant dexamethasone, however, the Deltasrs9 strain reactivated in the intestinal tissue after only 8 to 9 days, versus 2 weeks for the wild-type strain. Thus, SRS9 appears to play an important role in both persistence in the brain and reactivation in the intestine. Possible mechanisms for this are discussed.

  View details for DOI 10.1128/IAI.01862-06

  View details for Web of Science ID 000245421400009

  View details for PubMedID 17261600

  View details for PubMedCentralID PMC1865672

• Pulling together: an integrated model of Toxoplasma cell invasion. Current opinion in microbiology Carruthers, V., Boothroyd, J. C. 2007; 10 (1): 83-89

  Abstract

  The protozoan Toxoplasma gondii invades a wide array of animal cells using an actin/myosin-based motor complex to drive active penetration. This broad specificity implies that the parasite has developed a means of using a widely expressed receptor, many different receptors, or perhaps a receptor produced by T. gondii itself. Recently, there has been an explosion in identification of the molecules involved, including those that comprise the 'moving junction' that slides over the parasite as it invades. The emerging model is that invasion comprises at least seven steps that progressively increase the parasite's grip on the host surface, form the moving junction and enlist the motor complex to drive entry. These recent findings have led to new hypotheses regarding the parasite's broad host-specificity.

  View details for PubMedID 16837236

• Pulling together: an integrated model of Toxoplasma cell invasion CURRENT OPINION IN MICROBIOLOGY Carruthers, V. B., Boothroyd, J. C. 2007; 10 (1): 82-89

  View details for Web of Science ID 000244809900014

• Infection with Toxoplasma gondii bradyzoites has a diminished impact on host transcript levels relative to tachyzoite infection INFECTION AND IMMUNITY Fouts, A. E., Boothroyd, J. C. 2007; 75 (2): 634-642

  Abstract

  Toxoplasma gondii, an intracellular pathogen, has the potential to infect nearly every warm-blooded animal but rarely causes morbidity. The ability for the parasite to convert to the bradyzoite stage and live inside slow-growing cysts that can go unnoticed by the host immune system allows for parasite persistence for the life of the infected host. This intracellular survival likely necessitates host cell modulation, and tachyzoites are known to modify a number of signaling cascades within the host to promote parasite survival. Little is known, however, about how bradyzoites manipulate their host cell. Microarrays were used to profile the host transcriptional changes caused by bradyzoite infection and compared to those of tachyzoite-infected and uninfected hosts cells 2 days postinfection in vitro. Infection resulted in chemokine, cytokine, extracellular matrix, and growth factor transcript level changes. A small group of genes were specifically induced by tachyzoite infection, including granulocyte-macrophage colony-stimulating factor, BCL2-related protein A1, and interleukin-24. Bradyzoite infection yielded only about half the changes seen with tachyzoite infection, and those changes that did occur were almost all of lower magnitude than those induced by tachyzoites. These results suggest that bradyzoites lead a more stealthy existence within the infected host cell.

  View details for DOI 10.1128/IAI.01228-06

  View details for Web of Science ID 000243865500010

  View details for PubMedID 17088349

  View details for PubMedCentralID PMC1828502

• Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue NATURE Saeij, J. P., Coller, S., Boyle, J. P., Jerome, M. E., White, M. W., Boothroyd, J. C. 2007; 445 (7125): 324-327

  Abstract

  Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, can cause severe disease in humans with an immature or suppressed immune system. The outcome of Toxoplasma infection is highly dependent on the strain type, as are many of its in vitro growth properties. Here we use genetic crosses between type II and III lines to show that strain-specific differences in the modulation of host cell transcription are mediated by a putative protein kinase, ROP16. Upon invasion by the parasite, this polymorphic protein is released from the apical organelles known as rhoptries and injected into the host cell, where it ultimately affects the activation of signal transducer and activator of transcription (STAT) signalling pathways and consequent downstream effects on a key host cytokine, interleukin (IL)-12. Our findings provide a new mechanism for how an intracellular eukaryotic pathogen can interact with its host and reveal important differences in how different Toxoplasma lineages have evolved to exploit this interaction.

  View details for DOI 10.1038/nature05395

  View details for Web of Science ID 000243504700047

  View details for PubMedID 17183270

  View details for PubMedCentralID PMC2637441

• Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of a infected host cells EUKARYOTIC CELL Gilbert, L. A., Ravindran, S., Turetzky, J. M., Boothroyd, J. C., Bradley, P. J. 2007; 6 (1): 73-83

  Abstract

  Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.

  View details for DOI 10.1128/EC.00309-06

  View details for Web of Science ID 000244895400006

  View details for PubMedID 17085638

• Polymorphic secreted kinases are key virulence factors in toxoplasmosis SCIENCE Saeij, J. P., Boyle, J. P., Coller, S., Taylor, S., Sibley, L. D., Brooke-Powell, E. T., Ajioka, J. W., Boothroyd, J. C. 2006; 314 (5806): 1780-1783

  Abstract

  The majority of known Toxoplasma gondii isolates from Europe and North America belong to three clonal lines that differ dramatically in their virulence, depending on the host. To identify the responsible genes, we mapped virulence in F(1) progeny derived from crosses between type II and type III strains, which we introduced into mice. Five virulence (VIR) loci were thus identified, and for two of these, genetic complementation showed that a predicted protein kinase (ROP18 and ROP16, respectively) is the key molecule. Both are hypervariable rhoptry proteins that are secreted into the host cell upon invasion. These results suggest that secreted kinases unique to the Apicomplexa are crucial in the host-pathogen interaction.

  View details for DOI 10.1126/science.1133690

  View details for Web of Science ID 000242833600060

  View details for PubMedID 17170306

  View details for PubMedCentralID PMC2646183

• Just one cross appears capable of dramatically altering the population biology of a eukaryotic pathogen like Toxoplasma gondii PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boyle, J. P., Rajasekar, B., Saeij, J. P., Ajiokat, J. W., Berriman, M., Paulsen, I., Roos, D. S., Sibley, L. D., White, M. W., Boothroyd, J. C. 2006; 103 (27): 10514-10519

  Abstract

  Toxoplasma gondii, an obligate intracellular protozoan of the phylum Apicomplexa, is estimated to infect over a billion people worldwide as well as a great many other mammalian and avian hosts. Despite this ubiquity, the vast majority of human infections in Europe and North America are thought to be due to only three genotypes. Using a genome-wide analysis of single-nucleotide polymorphisms, we have constructed a genealogy for these three lines. The data indicate that types I and III are second- and first-generation offspring, respectively, of a cross between a type II strain and one of two ancestral strains. An extant T. gondii strain (P89) appears to be the modern descendant of the non-type II parent of type III, making the full genealogy of the type III clonotype known. The simplicity of this family tree demonstrates that even a single cross can lead to the emergence and dominance of a new clonal genotype that completely alters the population biology of a sexual pathogen.

  View details for DOI 10.1073/pnas.0510319103

  View details for Web of Science ID 000239069400065

  View details for PubMedID 16801557

  View details for PubMedCentralID PMC1502489

• Plasmodium falciparum AMA1 binds a rhoptry neck protein homologous to TgRON4, a component of the moving junction in Toxoplasma gondii EUKARYOTIC CELL Alexander, D. L., Arastu-Kapur, S., Dubremetz, J., Boothroyd, J. C. 2006; 5 (7): 1169-1173

  Abstract

  Plasmodium falciparum apical membrane antigen 1 (PfAMA1) coimmunoprecipitates with the Plasmodium homologue of TgRON4, a secreted rhoptry neck protein of Toxoplasma gondii that migrates at the moving junction in association with TgAMA1 during invasion. PfRON4 also originates in the rhoptry necks, suggesting that this unusual collaboration of micronemes and rhoptries is a conserved feature of Apicomplexa.

  View details for DOI 10.1128/EC.00040-06

  View details for Web of Science ID 000239155700018

  View details for PubMedID 16835460

  View details for PubMedCentralID PMC1489286

• Analysis of gene expression during development: Lessons from the Apicomplexa MICROBES AND INFECTION Boyle, J. P., Saeij, J. P., Cleary, M. D., Boothroyd, J. C. 2006; 8 (6): 1623-1630

  Abstract

  Apicomplexans are responsible for significant human and animal disease worldwide, including malaria and toxoplasmosis. Herein we summarize recent advances in gene expression analysis in these eukaryotic pathogens, especially with respect to their developmental biology, and discuss the impact this work may have on the development of new vaccines and chemotherapeutics.

  View details for DOI 10.1016/j.micinf.2005.11.017

  View details for Web of Science ID 000239253100023

  View details for PubMedID 16697685

• Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space CELL Coppens, I., Dunn, J. D., Romano, J. D., Pypaert, M., Zhang, H., Boothroyd, J. C., Joiner, K. A. 2006; 125 (2): 261-274

  Abstract

  The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

  View details for DOI 10.1016/j.cell.2006.01.056

  View details for Web of Science ID 000237241500017

  View details for PubMedID 16630815

• A novel rhoptry protein in Toxoplasma gondii bradyzoites and merozoites MOLECULAR AND BIOCHEMICAL PARASITOLOGY Schwarz, J. A., Fouts, A. E., Cummings, C. A., Ferguson, D. J., Boothroyd, J. C. 2005; 144 (2): 159-166

  Abstract

  The secretory organelles of Toxoplasma gondii orchestrate invasion of the host cell and establish the parasitophorous vacuole. Although much has been learned about the roles played by these organelles in invasion by the tachyzoite stage, little is known about the contents or functions of these organelles during bradyzoite development or pathogenesis. We identified a novel protein that localizes to the rhoptries of the bradyzoite stage, but is absent from the tachyzoite stage. This protein, BRP1, first appears in the nascent rhoptries during the first division of bradyzoite stage development. We observed secretion of BRP1 and other rhoptry proteins into the parasitophorous vacuole during bradyzoite development in vitro, but there was no evidence that this occurs in vivo. Brp1 knockout parasites did not appear to have any developmental or growth defects in vitro, and were able to establish infections in mice both as tachyzoites (via intraperitoneal injection of in vitro-derived tachyzoites) or bradyzoites (via oral gavage using cysts harvested from mouse brain). Mice infected using brain cysts from the brp1 knockout or the control strain developed similar numbers and sizes of brain cysts. Thus BRP1 does not appear to play an essential role in development of the bradyzoite stage, development of brain cysts, or oral infection of new hosts, at least in the mouse model used here. Since we also observed that BRP1 is expressed in the merozoite stages in the gut of infected cats, the coccidian phase of the life cycle may be where BRP1 plays its most important role.

  View details for DOI 10.1016/j.molbiopara.2005.08.011

  View details for Web of Science ID 000233713400004

  View details for PubMedID 16182390

• Proteomic analysis of rhoptry organelles reveals many novel constituents for host-parasite interactions in Toxoplasma gondii JOURNAL OF BIOLOGICAL CHEMISTRY Bradley, P. J., Ward, C., Cheng, S. J., Alexander, D. L., Coller, S., Coombs, G. H., Dunn, J. D., Ferguson, D. J., Sanderson, S. J., Wastling, J. M., Boothroyd, J. C. 2005; 280 (40): 34245-34258

  Abstract

  Rhoptries are specialized secretory organelles that are uniquely present within protozoan parasites of the phylum Apicomplexa. These obligate intracellular parasites comprise some of the most important parasites of humans and animals, including the causative agents of malaria (Plasmodium spp.) and chicken coccidiosis (Eimeria spp.). The contents of the rhoptries are released into the nascent parasitophorous vacuole during invasion into the host cell, and the resulting proteins often represent the literal interface between host and pathogen. We have developed a method for highly efficient purification of rhoptries from one of the best studied Apicomplexa, Toxoplasma gondii, and we carried out a detailed proteomic analysis using mass spectrometry that has identified 38 novel proteins. To confirm their rhoptry origin, antibodies were raised to synthetic peptides and/or recombinant protein. Eleven of 12 of these yielded antibody that showed strong rhoptry staining by immunofluorescence within the rhoptry necks and/or their bulbous base. Hemagglutinin epitope tagging confirmed one additional novel protein as from the rhoptry bulb. Previously identified rhoptry proteins from Toxoplasma and Plasmodium were unique to one or the other organism, but our elucidation of the Toxoplasma rhoptry proteome revealed homologues that are common to both. This study also identified the first Toxoplasma genes encoding rhoptry neck proteins, which we named RONs, demonstrated that toxofilin and Rab11 are rhoptry proteins, and identified novel kinases, phosphatases, and proteases that are likely to play a key role in the ability of the parasite to invade and co-opt the host cell for its own survival and growth.

  View details for DOI 10.1074/jbc.M504158200

  View details for Web of Science ID 000232229700067

  View details for PubMedID 16002398

• Identification of the moving junction complex of Toxoplasma gondii: A collaboration between distinct secretory organelles PLOS PATHOGENS Alexander, D. L., Mital, J., Ward, G. E., Bradley, P., Boothroyd, J. C. 2005; 1 (2): 137-149

  Abstract

  Apicomplexan parasites, including Toxoplasma gondii and Plasmodium sp., are obligate intracellular protozoa. They enter into a host cell by attaching to and then creating an invagination in the host cell plasma membrane. Contact between parasite and host plasma membranes occurs in the form of a ring-shaped moving junction that begins at the anterior end of the parasite and then migrates posteriorly. The resulting invagination of host plasma membrane creates a parasitophorous vacuole that completely envelops the now intracellular parasite. At the start of this process, apical membrane antigen 1 (AMA1) is released onto the parasite surface from specialized secretory organelles called micronemes. The T. gondii version of this protein, TgAMA1, has been shown to be essential for invasion but its exact role has not previously been determined. We identify here a trio of proteins that associate with TgAMA1, at least one of which associates with TgAMA1 at the moving junction. Surprisingly, these new proteins derive not from micronemes, but from the anterior secretory organelles known as rhoptries and specifically, for at least two, from the neck portion of these club-shaped structures. Homologues for these AMA1-associated proteins are found throughout the Apicomplexa strongly suggesting that this moving junction apparatus is a conserved feature of this important class of parasites. Differences between the contributing proteins in different species may, in part, be the result of selective pressure from the different niches occupied by these parasites.

  View details for DOI 10.1371/journal.ppat.0010017

  View details for Web of Science ID 000202893800005

  View details for PubMedID 16244709

  View details for PubMedCentralID PMC1262624

• Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host TRENDS IN PARASITOLOGY Saeij, J. P., Boyle, J. P., Boothroyd, J. C. 2005; 21 (10): 476-481

  Abstract

  Toxoplasma gondii is one of the most successful protozoan parasites owing to its ability to manipulate the immune system and establish a chronic infection. There are many T. gondii strains but the majority identified in Europe and North America falls into three distinct clonal lineages. Many studies have investigated the ability of T. gondii to manipulate its host but few have examined directly whether the three lineages differ in this ability.

  View details for DOI 10.1016/j.pt.10.1016/j.pt.2005.08.001

  View details for Web of Science ID 000232443000010

  View details for PubMedID 16098810

• Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-to-bradyzoite differentiation in Toxoplasma gondii INFECTION AND IMMUNITY Vanchinathan, P., Brewer, J. L., Harb, O. S., Boothroyd, J. C., Singh, U. 2005; 73 (10): 6680-6688

  Abstract

  During its life cycle in intermediate hosts, Toxoplasma gondii exists in two interconverting developmental stages: tachyzoites and bradyzoites. This interconversion is essential for the survival and pathogenicity of the parasite, but little is known about the genetic mechanisms that control this process. We have previously generated tachyzoite-to-bradyzoite differentiation (Tbd(-)) mutants using chemical mutagenesis and a green fluorescent protein-based selection strategy. The genetic loci responsible for the Tbd(-) phenotype, however, could not be identified. We have now used an insertional mutagenesis strategy to generate two differentiation mutants: TBD-5 and TBD-6 that switch to bradyzoites at 10 and 50% of wild-type levels, respectively. In TBD-6 there is a single insertion of the mutagenesis vector 164 bp upstream of the transcription start site of a gene encoding a zinc finger protein (ZFP1). Disruption of this locus in wild-type parasites reproduces the decreased stage conversion phenotype. ZFP1 is targeted to the parasite nucleolus by CCHC motifs and significantly altered expression levels are toxic to the parasites. This represents the first identification of a gene necessary for efficient conversion of tachyzoites to bradyzoites.

  View details for DOI 10.1128/IAI.73.10.6680-6688.2005

  View details for Web of Science ID 000232087600055

  View details for PubMedID 16177345

  View details for PubMedCentralID PMC1230886

• Stage-specific expression of surface antigens by Toxoplasma gondii as a mechanism to facilitate parasite persistence JOURNAL OF IMMUNOLOGY Kim, S. K., Boothroyd, J. C. 2005; 174 (12): 8038-8048

  Abstract

  Toxoplasma persists in the face of a functional immune system. This success critically depends on the ability of parasites to activate a strong adaptive immune response during acute infection with tachyzoites that eliminates most of the parasites and to undergo stage conversion to bradyzoites that encyst and persist predominantly in the brain. A dramatic change in antigenic composition occurs during stage conversion, such that tachyzoites and bradyzoites express closely related but antigenically distinct sets of surface Ags belonging to the surface Ag 1 (SAG1)-related sequence (SRS) family. To test the contribution of this antigenic switch to parasite persistence, we engineered parasites to constitutively express the normally bradyzoite-specific SRS9 (SRS9(c)) mutants and tachyzoite-specific SAG1 (SAG1(c)) mutants. SRS9(c) but not wild-type parasites elicited a SRS9-specific immune response marked by IFN-gamma production, suggesting that stage-specificity of SRS Ags determines their immunogenicity in infection. The induction of a SRS9-specific immune response correlated with a continual decrease in the number of SRS9(c) cysts persisting in the brain. In contrast, SAG1(c) mutants produced reduced brain cyst loads early in chronic infection, but these substantially increased over time accompanying a hyperproduction of IFN-gamma, TNF-alpha, and IL-10, and severe encephalitis. We conclude that stage-specific expression of SRS Ags is among the key mechanisms by which optimal parasite persistency is established and maintained.

  View details for Web of Science ID 000229791400069

  View details for PubMedID 15944311

• Identification and disruption of a rhoptry-localized homologue of sodium hydrogen exchangers in Toxoplasma gondii INTERNATIONAL JOURNAL FOR PARASITOLOGY Karasov, A. O., Boothroyd, J. C., Arrizabalaga, G. 2005; 35 (3): 285-291

  Abstract

  Na+/H+ exchangers (NHEs) are ubiquitous membrane proteins that catalyze the exchange of Na+ for H+ and are critical in pH and cell volume regulation, as well as osmotolerance. In this study, we identify and characterize a novel NHE, TgNHE2, in Toxoplasma gondii. Immunofluorescence studies show that TgNHE2 is localized to the rhoptries, secretory organelles involved in invasion. TgNHE2 is the first intracellular NHE to be characterized in a protozoan parasite and its localization suggests possible roles for the rhoptries in osmotolerance and/or as secretory lysosomes-like granules.

  View details for DOI 10.1016/j.ijpara.2004.11.015

  View details for Web of Science ID 000227605300005

  View details for PubMedID 15722080

• Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay NATURE BIOTECHNOLOGY Cleary, M. D., Meiering, C. D., Jan, E., Guymon, R., Boothroyd, J. C. 2005; 23 (2): 232-237

  Abstract

  Standard microarrays measure mRNA abundance, not mRNA synthesis, and therefore cannot identify the mechanisms that regulate gene expression. We have developed a method to overcome this limitation by using the salvage enzyme uracil phosphoribosyltransferase (UPRT) from the protozoan Toxoplasma gondii. T. gondii UPRT has been well characterized because of its application in monitoring parasite growth: mammals lack this enzyme activity and thus only the parasite incorporates (3)H-uracil into its nucleic acids. In this study we used RNA labeling by UPRT to determine the roles of mRNA synthesis and decay in the control of gene expression during T. gondii asexual development. We also used this approach to specifically label parasite RNA during a mouse infection and to incorporate thio-substituted uridines into the RNA of human cells engineered to express T. gondii UPRT, indicating that engineered UPRT expression will allow cell-specific analysis of gene expression in organisms other than T. gondii.

  View details for DOI 10.1038/nbt1061

  View details for Web of Science ID 000226797600031

  View details for PubMedID 15685165

• Bioluminescence imaging of Toxoplasma gondii infection in living mice reveals dramatic differences between strains INFECTION AND IMMUNITY Saeij, J. P., Boyle, J. P., Grigg, M. E., Arrizabalaga, G., Boothroyd, J. C. 2005; 73 (2): 695-702

  Abstract

  We examined the in vivo growth, dissemination, and reactivation of strains of the protozoan parasite Toxoplasma gondii using a bioluminescence-based imaging system. Two T. gondii strains, one with a highly virulent disease phenotype in mice (S23) and the other with a 1,000-fold-lower virulence phenotype (S22), were engineered to stably express the light-emitting protein luciferase. One clone of each wild-type strain was isolated, and the two clones (S23-luc7 and S22-luc2) were found to express similar levels of luciferase. Mice were infected intraperitoneally with S23-luc7 (50 or 5 parasites) or S22-luc2 (500, 50, or 5 parasites), and the progress of the infections was examined noninvasively following injection of the substrate for luciferase, d-luciferin. In mice infected with 50 S23-luc7 parasites, the parasites grew exponentially within the peritoneal cavity (as measured by light emitted from luciferase-expressing parasites) during days 1 to 10 p.i., and this proliferation continued until there was severe disease. In mice infected with 500 S22-luc2 parasites, the parasites proliferated in a fashion similar to the S23-luc7 proliferation during days 1 to 6, but this was followed by a precipitous drop in the signal to levels below the limit of detection. Using this technique, we were also able to observe the process of reactivation of T. gondii in chronically infected mice. After treatment with dexamethasone, we detected reactivation of toxoplasmosis in mice infected with S23-luc7 and S22-luc2. During reactivation, growth of S23-luc7 was initially detected primarily in the head and neck area, while in S22-luc2-infected mice the parasites were detected primarily in the abdomen. This method has great potential for identifying important differences in the dissemination and growth of different T. gondii strains, especially strains with dramatically different disease outcomes.

  View details for DOI 10.1128/IAI.73.2.695-702.2005

  View details for Web of Science ID 000226731700004

  View details for PubMedID 15664907

  View details for PubMedCentralID PMC547072

• Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii NUCLEIC ACIDS RESEARCH Khan, A., Taylor, S., Su, C., Mackey, A. J., BOYLE, J., Cole, R., Glover, D., Tang, K., Paulsen, I. T., Berriman, M., Boothroyd, J. C., Pfefferkorn, E. R., Dubey, J. P., Ajioka, J. W., Roos, D. S., Wootton, J. C., Sibley, L. D. 2005; 33 (9): 2980-2992

  Abstract

  Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.

  View details for DOI 10.1093/nar/gki604

  View details for Web of Science ID 000229544600029

  View details for PubMedID 15911631

• A GFP-based motif-trap reveals a novel mechanism of targeting for the Toxoplasma ROP4 protein MOLECULAR AND BIOCHEMICAL PARASITOLOGY Bradley, P. J., Li, N., Boothroyd, J. C. 2004; 137 (1): 111-120

  Abstract

  The protozoan parasite Toxoplasma gondii is a highly specialized eukaryote that contains a remarkable number of intracellular compartments, some unique to Apicomplexans and others typical of eukaryotes in general. We have established a green fluorescent protein (GFP)-based motif-trap to identify proteins targeted to different intracellular locations and subsequently the signals responsible for this sorting. The motif-trap involves the transfection and integration of a linearized GFP construct which lacks a promoter and an initiator methionine codon. FACS is used to isolate parasites in which GFP fuses in-frame into a coding region followed by screening by fluorescence microscopy for those containing GFP targeted to specific intracellular compartments. GFP trapping was successful using vectors designed for integration into regions encoding exons and vectors that were engineered with a splice acceptor site for integration into regions encoding introns. This strategy differs from most protein traps in that the resulting fusions are expressed from the endogenous promoter and starting methionine. Thus, problems from inappropriate expression levels or the creation of fortuitous targeting signals seen in library-based traps are diminished. Using this approach, we have trapped GFP localized to a number of intracellular compartments including the nucleus, nucleolus, endoplasmic reticulum, cytosol, parasite surface and rhoptries of Toxoplasma. Further analysis of a parasite clone containing GFP targeted to the rhoptries shows GFP fused to the gene encoding the rhoptry protein ROP4 and has elucidated an additional mechanism for targeting of this protein.

  View details for DOI 10.1016/j.molbiopara.2004.05.003

  View details for Web of Science ID 000223376800012

  View details for PubMedID 15279957

• The induction of acute ileitis by a single microbial antigen of Toxoplasma gondii JOURNAL OF IMMUNOLOGY Rachinel, N., Buzoni-Gatel, D., Dutta, C., Mennechet, F. J., Luangsay, S., Minns, L. A., Grigg, M. E., Tomavo, S., Boothroyd, J. C., Kasper, L. H. 2004; 173 (4): 2725-2735

  Abstract

  The role of specific microbial Ags in the induction of experimental inflammatory bowel disease is poorly understood. Oral infection of susceptible C57BL/6 mice with Toxoplasma gondii results in a lethal ileitis within 7-9 days postinfection. An immunodominant Ag of T. gondii (surface Ag 1 (SAG1)) that induces a robust B and T cell-specific response has been identified and a SAG1-deficient parasite (Deltasag1) engineered. We investigated the ability of Deltasag1 parasite to induce a lethal intestinal inflammatory response in susceptible mice. C57BL/6 mice orally infected with Deltasag1 parasites failed to develop ileitis. In vitro, the mutant parasites replicate in both enterocytes and dendritic cells. In vivo, infection with the mutant parasites was associated with a decrease in the chemokine and cytokine production within several compartments of the gut-associated cell population. RAG-deficient (RAG1(-/-)) mice are resistant to the development of the ileitis after T. gondii infection. Adoptive transfer of Ag-specific CD4(+) effector T lymphocytes isolated from C57BL/6-infected mice into RAG(-/-) mice conferred susceptibility to the development of the intestinal disease. In contrast, CD4(+) effector T lymphocytes from mice infected with the mutant Deltasag1 strain failed to transfer the pathology. In addition, resistant mice (BALB/c) that fail to develop ileitis following oral infection with T. gondii were rendered susceptible following intranasal presensitization with the SAG1 protein. This process was associated with a shift toward a Th1 response. These findings demonstrate that a single Ag (SAG1) of T. gondii can elicit a lethal inflammatory process in this experimental model of pathogen-driven ileitis.

  View details for Web of Science ID 000223208900063

  View details for PubMedID 15294991

• Ionophore-resistant mutant of Toxoplasma gondii reveals involvement of a sodium/hydrogen exchanger in calcium regulation JOURNAL OF CELL BIOLOGY Arrizabalaga, G., RUIZ, F., Moreno, S., Boothroyd, J. C. 2004; 165 (5): 653-662

  Abstract

  Calcium is a critical mediator of many intracellular processes in eukaryotic cells. In the obligate intracellular parasite Toxoplasma gondii, for example, a rise in [Ca2+] is associated with significant morphological changes and rapid egress from host cells. To understand the mechanisms behind such dramatic effects, we isolated a mutant that is altered in its responses to the Ca2+ ionophore A23187 and found the affected gene encodes a homologue of Na+/H+ exchangers (NHEs) located on the parasite's plasma membrane. We show that in the absence of TgNHE1, Toxoplasma is resistant to ionophore-induced egress and extracellular death and amiloride-induced proton efflux inhibition. In addition, the mutant has increased levels of intracellular Ca2+, which explains its decreased sensitivity to A23187. These results provide direct genetic evidence of a role for NHE1 in Ca2+ homeostasis and important insight into how this ubiquitous pathogen senses and responds to changes in its environment.

  View details for DOI 10.1083/jcb.200309097

  View details for Web of Science ID 000221986200007

  View details for PubMedID 15173192

  View details for PubMedCentralID PMC2172388

• An unusual genotype of Toxoplasma gondii is common in California sea otters (Enhydra lutris nereis) and is a cause of mortality 7th International Congress on Toxoplasmosis Miller, M. A., Grigg, M. E., Kreuder, C., James, E. R., Melli, A. C., Crosbie, P. R., Jessup, D. A., Boothroyd, J. C., Brownstein, D., Conrad, P. A. ELSEVIER SCI LTD. 2004: 275–84

  Abstract

  Toxoplasma gondii-associated meningoencephalitis is a significant disease of California sea otters (Enhydra lutris nereis), responsible for 16% of total mortality in fresh, beachcast carcasses. Toxoplasma gondii isolates were obtained from 35 California otters necropsied between 1998 and 2002. Based on multi-locus PCR-restriction fragment length polymorphism and DNA sequencing at conserved genes (18S rDNA, ITS-1) and polymorphic genes (B1, SAG1, SAG3 and GRA6), two distinct genotypes were identified: type II and a novel genotype, here called type x, that possessed distinct alleles at three of the four polymorphic loci sequenced. The majority (60%) of sea otter T. gondii infections were of genotype x, with the remaining 40% being of genotype II. No type I or III genotypes were identified. Epidemiological methods were used to examine the relationship between isolated T. gondii genotype(s) and spatial and demographic risk factors, such as otter stranding location and sex, as well as specific outcomes related to pathogenicity, such as severity of brain inflammation on histopathology and T. gondii-associated mortality. Differences were identified with respect to T. gondii genotype and sea otter sex and stranding location along the California coast. Localised spatial clustering was detected for both type II (centred within Monterey Bay) and x (centred near Morro Bay)-infected otters. The Morro Bay cluster of type x-infected otters overlaps previously reported high-risk areas for sea otter infection and mortality due to T. gondii. Nine of the 12 otters that had T. gondii-associated meningoencephalitis as a primary cause of death were infected with type x parasites.

  View details for DOI 10.1016/j.ijpara.2003.12.008

  View details for Web of Science ID 000220413500004

  View details for PubMedID 15003489

• Role of calcium during Toxoplasma gondii invasion and egress 7th International Congress on Toxoplasmosis Arrizabalaga, G., Boothroyd, J. C. ELSEVIER SCI LTD. 2004: 361–68

  Abstract

  Calcium is a ubiquitous signalling molecule involved in a large number of cellular processes in eukaryotic cells. In the obligate intracellular parasite, Toxoplasma gondii, for example, a rise in calcium concentration is associated with significant morphological changes, secretion of proteins involved in host cell invasion and rapid egress from the host cell. Recent findings indicate that calcium released from the parasite's intracellular pools is necessary and sufficient to induce some of the events critical for invasion and egress. In addition, ethanol, a powerful inducer of invasion-related events, is shown here to also induce rapid egress from the host cell, indicating that a common mechanism for calcium release might be involved during both invasion and egress.

  View details for DOI 10.1016/j.ijpara.2003.11.017

  View details for Web of Science ID 000220413500011

  View details for PubMedID 15003496

• DNA microarrays in parasitology: strengths and limitations TRENDS IN PARASITOLOGY Boothroyd, J. C., Blader, I., Cleary, M., Singh, U. 2003; 19 (10): 470-476

  Abstract

  Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced and where little, if any, annotation is available. The focus of this review is on how to use and apply microarrays to these situations.

  View details for DOI 10.1016/j.pt.2003.08.002

  View details for Web of Science ID 000186058400012

  View details for PubMedID 14519585

• Serotyping of Toxoplasma gondii infections in humans using synthetic peptides JOURNAL OF INFECTIOUS DISEASES Kong, J. T., Grigg, M. E., Uyetake, L., Parmley, S., Boothroyd, J. C. 2003; 187 (9): 1484-1495

  Abstract

  To determine whether the characteristics of disease due to Toxoplasma gondii (toxoplasmosis) are dependent on the infecting strain, we have developed an enzyme-linked immunosorbent assay for typing strains that uses infection serum reacted against polymorphic peptides derived from Toxoplasma antigens SAG2A, GRA3, GRA6, and GRA7. Pilot studies with infected mice established the validity of the approach, which was then tested with human serum. In 8 patients who had Sabin-Feldman dye test titers >64 and for whom the infecting strain type was known, the peptides correctly distinguished type II from non-type II infections. ELISA analysis of a second group of 10 infected pregnant women from whom the parasite strain had not been isolated gave a clear prediction of the strain type causing infection. This method should allow statistically significant data to be obtained about whether different strain types cause disease with different characteristics.

  View details for Web of Science ID 000182273700017

  View details for PubMedID 12717631

• Unprocessed Toxoplasma ROP1 is effectively targeted and secreted into the nascent parasitophorous vacuole MOLECULAR AND BIOCHEMICAL PARASITOLOGY Bradley, P. J., Hsieh, C. L., Boothroyd, J. C. 2002; 125 (1-2): 189-193

  View details for Web of Science ID 000179862000018

  View details for PubMedID 12467986

• Structure of the immunodominant surface antigen from the Toxoplasma gondii SRS superfamily NATURE STRUCTURAL BIOLOGY He, X. L., Grigg, M. E., Boothroyd, J. C., Garcia, K. C. 2002; 9 (8): 606-611

  Abstract

  Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrate. The surface of Toxoplasma is coated with a family of developmentally regulated glycosylphosphatidylinositol (GPI)-linked proteins (SRSs), of which SAG1 is the prototypic member. SRS proteins mediate attachment to host cells and interface with the host immune response to regulate the virulence of the parasite. The 1.7 A structure of the immunodominant SAG1 antigen reveals a homodimeric configuration in which the dimeric interface is mediated by an extended beta-sheet that forms a deep groove lined with positively charged amino acids. This basic groove seems to be conserved among SRS proteins and potentially serves as a sulfated proteoglycan-binding site on target cell surfaces, thus rationalizing the promiscuous attachment properties of Toxoplasma to a broad range of host cell types.

  View details for DOI 10.1038/nsb819

  View details for Web of Science ID 000177214200014

  View details for PubMedID 12091874

• Population biology of Toxoplasma gondii and its relevance to human infection: do different strains cause different disease? CURRENT OPINION IN MICROBIOLOGY Boothroyd, J. C., Grigg, M. E. 2002; 5 (4): 438-442

  Abstract

  Toxoplasma gondii is a protozoan parasite that is globally widespread and causes a common infection of many warm-blooded animals. It has an unusual population structure with a few clonally reproducing strains apparently dominating in many of its hosts, which include humans. In mice, the various strains of the parasite differ enormously in their virulence and disease presentation. In humans, disease manifestations are highly variable, ranging from asymptomatic to severe, especially in cases of brain and eye infection. Recent data suggest that, as with mice, at least part of this variability in human infection may be tied to the type of strain that causes the infection. Improvements in our knowledge of this parasite's population biology and ways to determine the genotype of an infecting strain should make it possible to test this relationship in various disease scenarios. Clear correlations will substantially affect the management of human disease, matching an aggressive infection with an equally aggressive treatment.

  View details for Web of Science ID 000177714300015

  View details for PubMedID 12160866

• Toxoplasma gondii asexual development: Identification of developmentally regulated genes and distinct patterns of gene expression EUKARYOTIC CELL Cleary, M. D., Singh, U., Blader, I. J., Brewer, J. L., Boothroyd, J. C. 2002; 1 (3): 329-340

  Abstract

  Asexual development in Toxoplasma gondii is a vital aspect of the parasite's life cycle, allowing transmission and avoidance of the host immune response. Differentiation of rapidly dividing tachyzoites into slowly growing, encysted bradyzoites involves significant changes in both physiology and morphology. We generated microarrays of approximately 4,400 Toxoplasma cDNAs, representing a minimum of approximately 600 genes (based on partial sequencing), and used these microarrays to study changes in transcript levels during tachyzoite-to-bradyzoite differentiation. This approach has allowed us to (i) determine expression profiles of previously described developmentally regulated genes, (ii) identify novel developmentally regulated genes, and (iii) identify distinct classes of genes based on the timing and magnitude of changes in transcript levels. Whereas microarray analysis typically involves comparisons of mRNA levels at different time points, we have developed a method to measure relative transcript abundance between genes at a given time point. This method was used to determine transcript levels in parasites prior to differentiation and to further classify bradyzoite-induced genes, thus allowing a more comprehensive view of changes in gene expression than is provided by standard expression profiles. Newly identified developmentally regulated genes include putative surface proteins (a SAG1-related protein, SRS9, and a mucin-domain containing protein), regulatory and metabolic enzymes (methionine aminopeptidase, oligopeptidase, aminotransferase, and glucose-6-phosphate dehydrogenase homologues), and a subset of genes encoding secretory organelle proteins (MIC1, ROP1, ROP2, ROP4, GRA1, GRA5, and GRA8). This analysis permits the first in-depth look at changes in gene expression during development of this complex protozoan parasite.

  View details for DOI 10.1128/EC.1.3.329-340.2002

  View details for Web of Science ID 000178729300002

  View details for PubMedID 12455982

  View details for PubMedCentralID PMC118016

• Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction MOLECULAR MICROBIOLOGY Singh, U., Brewer, J. L., Boothroyd, J. C. 2002; 44 (3): 721-733

  Abstract

  Developmental switching in Toxoplasma gondii, from the virulent tachyzoite to the relatively quiescent bradyzoite stage, is responsible for disease propagation and reactivation. We have generated tachyzoite to bradyzoite differentiation (Tbd-) mutants in T. gondii and used these in combination with a cDNA microarray to identify developmental pathways in bradyzoite formation. Four independently generated Tbd- mutants were analysed and had defects in bradyzoite development in response to multiple bradyzoite-inducing conditions, a stable phenotype after in vivo passages and a markedly reduced brain cyst burden in a murine model of chronic infection. Transcriptional profiles of mutant and wild-type parasites, growing under bradyzoite conditions, revealed a hierarchy of developmentally regulated genes, including many bradyzoite-induced genes whose transcripts were reduced in all mutants. A set of non-developmentally regulated genes whose transcripts were less abundant in Tbd- mutants were also identified. These may represent genes that mediate downstream effects and/or whose expression is dependent on the same transcription factors as the bradyzoite-induced set. Using these data, we have generated a model of transcription regulation during bradyzoite development in T. gondii. Our approach shows the utility of this system as a model to study developmental biology in single-celled eukaryotes including protozoa and fungi.

  View details for Web of Science ID 000175466900011

  View details for PubMedID 11994153

• An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii MOLECULAR MICROBIOLOGY Camps, M., Arrizabalaga, G., Boothroyd, J. 2002; 43 (5): 1309-1318

  Abstract

  Toxoplasma gondii is a protozoan sensitive to several inhibitors of prokaryotic translation (e.g. clindamycin, macrolides and tetracyclines). A priori, two prokaryotic-like organelles, the 'apicoplast' (a non-photosynthetic plastid) and the mitochondrion, are likely targets for these drugs. Without using overt mutagenesis, we selected two independent clones (ClnR-4 and ClnR-21) with strong and stable clindamycin resistance. Several lines with substantial but lower levels of resistance were also isolated with (XR-46) or without (ClnR-23) overt mutagenesis. The ClnR-4 and ClnR-21 mutants uniquely possess a G-->U point mutation at position 1857 of the apicoplast large-subunit rRNA, whereas no mutation was identified in this region for ClnR-23 or XR-46. Position 1857 corresponds to position 2061 in Escherichia coli where it is predicted to bind clindamycin. The mutation is present in all the apicoplast rDNA copies (an estimated 12 per organelle), indicative of a strong selective advantage in the presence of clindamycin. In the absence of drug, however, such a mutation is unlikely to be neutral, as the G is a critical contributor to the transpeptidation reaction and absolutely conserved in all kingdoms. This may explain why ClnR-4 shows a slight growth defect in vitro. These mutants provide direct genetic evidence that apicoplast translation is the target for clindamycin in Toxoplasma. Further, their sensitivity profiles to other antibiotics specific for the large ribosomal subunit (macrolides and chloramphenicol) and, intriguingly, the small subunit (doxycycline) argue that these drugs also target the apicoplast ribosome.

  View details for Web of Science ID 000174774100020

  View details for PubMedID 11918815

• Immediate/early response to Trypanosoma cruzi infection involves minimal modulation of host cell transcription JOURNAL OF BIOLOGICAL CHEMISTRY de Avalos, S. V., Blader, I. J., Fisher, M., Boothroyd, J. C., Burleigh, B. A. 2002; 277 (1): 639-644

  Abstract

  Host cell infection by the intracellular pathogen, Trypanosoma cruzi, involves activation of signaling pathways, cytoskeletal reorganization, and targeted recruitment of host cell lysosomes. To determine the consequences of T. cruzi invasion on host cell gene expression, high density microarrays consisting of approximately 27,000 human cDNAs were hybridized with fluorescent probes generated from T. cruzi-infected human fibroblasts (HFF) at early time points following infection (2-24 h). Surprisingly, no genes were induced > or =2-fold in HFF between 2 and 6 h post-infection (hpi) in repeated experiments while immediate repression of six host cell transcripts was observed. A significant increase in transcript abundance for 106 host cell genes was observed at 24 hpi. Among the most highly induced is a set of interferon-stimulated genes, indicative of a type I interferon (IFN) response to T. cruzi. In support of this, T. cruzi-infected fibroblasts begin to secrete IFNbeta at 18 hpi following the induction of IFNbeta transcripts. As compared with global transcriptional responses evoked by other intracellular pathogens, T. cruzi is a stealth parasite that elicits few changes in host cell transcription during the initiation of infection.

  View details for DOI 10.1074/jbc.M109037200

  View details for Web of Science ID 000173087900084

  View details for PubMedID 11668183

• Success and virulence in toxoplasma as the result of sexual recombination between two distinct ancestries SCIENCE Grigg, M. E., Bonnefoy, S., Hehl, A. B., Suzuki, Y., Boothroyd, J. C. 2001; 294 (5540): 161-165

  Abstract

  Toxoplasma gondii is a common human pathogen causing serious, even fatal, disease in the developing fetus and in immunocompromised patients. Despite its ability to reproduce sexually and its broad geographic and host range, Toxoplasma has a clonal population structure comprised principally of three lines. We have analyzed 15 polymorphic loci in the archetypal type I, II, and III strains and found that polymorphism was limited to, at most, two rather than three allelic classes and no polymorphism was detected between alleles in strains of a given type. Multilocus analysis of 10 nonarchetypal isolates likewise clustered the vast majority of alleles into the same two distinct ancestries. These data strongly suggest that the currently predominant genotypes exist as a pandemic outbreak from a genetic mixing of two discrete ancestral lines. To determine if such mixing could lead to the extreme virulence observed for some strains, we examined the F(1) progeny of a cross between a type II and III strain, both of which are relatively avirulent in mice. Among the progeny were recombinants that were at least 3 logs more virulent than either parent. Thus, sexual recombination, by combining polymorphisms in two distinct and competing clonal lines, can be a powerful force driving the natural evolution of virulence in this highly successful pathogen.

  View details for Web of Science ID 000171448800052

  View details for PubMedID 11588262

• Surface antigens of Toxoplasma gondii: variations on a theme INTERNATIONAL JOURNAL FOR PARASITOLOGY Lekutis, C., Ferguson, D. J., Grigg, M. E., Camps, M., Boothroyd, J. C. 2001; 31 (12): 1285-1292

  Abstract

  Toxoplasma gondii is an obligate intracellular protozoan parasite with an exceptionally broad host range. Recently, it has become apparent that the number of surface antigens (SAGs) it expresses may rival the number of genera it can infect. Most of these antigens belong to the developmentally regulated and distantly related SAG1 or SAG2 families. The genes encoding the surface antigens are distributed throughout the T. gondii genome, with remarkably little polymorphism observed at each locus. Results from a number of studies have suggested that the surface antigens play an important role in the biology of the parasite. For example, SAG3 null mutants generated by targeted disruption provide convincing evidence that this surface antigen, at least, functions during parasite attachment. Analyses of a SAG1 knockout in rodents, however, indicate that this surface antigen may play a crucial role in immune modulation or virulence attenuation. The current understanding of the SAG1 and SAG2 families will be discussed here.

  View details for Web of Science ID 000171427000002

  View details for PubMedID 11566296

• Targeted disruption of an essential RNA-binding protein perturbs cell division in Trypanosoma brucei MOLECULAR AND BIOCHEMICAL PARASITOLOGY Manger, I. D., Boothroyd, J. C. 2001; 116 (2): 239-245

  View details for Web of Science ID 000171004600015

  View details for PubMedID 11522358

• Unusual abundance of atypical strains associated with human ocular toxoplasmosis JOURNAL OF INFECTIOUS DISEASES Grigg, M. E., Ganatra, J., Boothroyd, J. C., Margolis, T. P. 2001; 184 (5): 633-639

  Abstract

  To facilitate genotyping of Toxoplasma gondii in vitreous fluid of patients with severe or atypical ocular toxoplasmosis, polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays were developed for SAG3 (p43) and SAG4 (p18), 2 single-copy surface antigen genes. Together with strategies for SAG1, SAG2, and B1, multilocus RFLP analyses were performed on PCR-amplified parasite DNA present in 12 clinical specimens. Most samples (8/12) were not infected by type II or type III mouse-avirulent strains. Only 1 type III and 3 type II strains were identified, all from immunosuppressed patients. In 6 otherwise healthy adults and in 1 immunosuppressed patient, the SAG1 allele associated with mouse virulence was amplified. Of 12 samples, 3 possessed true type I strains; 5 of 12 had new recombinant genotypes with alleles typical of type I or III strains at all loci examined. The unusual bias toward type I and/or recombinant genotypes bearing the SAG1 type I allele associated with mouse virulence in immunocompetent adults has important implications for the epidemiology and efficacious treatment of ocular toxoplasmosis.

  View details for Web of Science ID 000170429400016

  View details for PubMedID 11474426

• The pro region of Toxoplasma ROP1 is a rhoptry-targeting signal INTERNATIONAL JOURNAL FOR PARASITOLOGY Bradley, P. J., Boothroyd, J. C. 2001; 31 (11): 1177-1186

  Abstract

  The rhoptries of Toxoplasma gondii are regulated secretory organelles involved in the invasion of host cells. Rhoptry proteins are synthesised as pre-pro-proteins that are processed first to pro-proteins upon entrance into the secretory pathway, then processed again to their mature forms late in the secretory pathway. The pro-mature processing site of the rhoptry protein ROP1 has been determined, paving the way for understanding the role of the pro region in rhoptry protein function. We demonstrate here that the ROP1 pro region is sufficient for targeting a reporter protein (amino acids 34-471 of the Trypanosoma brucei VSG117 protein) to the rhoptries. These results, together with our previous work showing that rhoptry targeting is unaffected by deletion of the pro region, indicate that the ROP1 protein contains at least two signals that can function in rhoptry targeting.

  View details for Web of Science ID 000170803600003

  View details for PubMedID 11513886

• Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions MOLECULAR AND BIOCHEMICAL PARASITOLOGY Knoll, L. J., Furie, G. L., Boothroyd, J. C. 2001; 116 (1): 11-16

  Abstract

  The obligate intracellular parasite Toxoplasma gondii can infect virtually any nucleated cell in any warm-blooded host. Through the effort of many researchers, we are beginning to learn what makes T. gondii such a successful protozoan parasite. A high throughput genetic screen that allows simultaneous examination of a large panel of mutants would greatly facilitate a global investigation of this parasite. Signature-tagged mutagenesis uses a unique DNA sequence to tag an individual mutant so that it can later be identified within a pool. This system allows the efficient identification of parasites carrying mutations in genes that are essential for growth in restrictive but not permissive conditions. We have generated a bank of approximately 4900 signature-tagged T. gondii tachyzoites represented in 89 pools, each of which contains 60 uniquely tagged mutant parasites. We have demonstrated the usefulness of this negative screening strategy with a tissue culture model for pyrimidine salvage using resistance to the pro-drug FUDR. Mutants that are defective for growth in any defined growth condition versus standard tissue culture conditions can now be identified (eg, sensitive to a specific drug, growth in a specialized cell line, or growth within animals).

  View details for Web of Science ID 000170409300002

  View details for PubMedID 11463461

• Toxoplasma gondii: Selective killing of extracellular parasites by oxidation using pyrrolidine dithiocarbamate EXPERIMENTAL PARASITOLOGY Camps, M., Boothroyd, J. C. 2001; 98 (4): 206-214

  Abstract

  Extracellular Toxoplasma parasites are sensitive to pyrrolidine dithiocarbamate (PDTC) at low micromolar concentrations. Loss of parasite viability following PDTC treatment is shown to be mediated by oxidation, which is reminiscent of PDTC killing in mammalian cells. Intracellular parasites, by contrast, are resistant to PDTC killing, although treatment does cause reversible growth arrest. In addition to the possible implications relative to the biology of the parasite, these observations suggest that PDTC could be of use in eliminating undesired extracellular parasites during assays and selections in vitro.

  View details for DOI 10.1006/expr.2001.4636

  View details for Web of Science ID 000171260100004

  View details for PubMedID 11560413

• Microarray analysis reveals previously unknown changes in Toxoplasma gondii-infected human cells JOURNAL OF BIOLOGICAL CHEMISTRY Blader, I. J., Manger, I. D., Boothroyd, J. C. 2001; 276 (26): 24223-24231

  Abstract

  Cells infected with the intracellular protozoan parasite Toxoplasma gondii undergo up-regulation of pro-inflammatory cytokines, organelle redistribution, and protection from apoptosis. To examine the molecular basis of these and other changes, gene expression profiles of human foreskin fibroblasts infected with Toxoplasma were studied using human cDNA microarrays consisting of approximately 22,000 known genes and uncharacterized expressed sequence tags. Early during infection (1-2 h), <1% of all genes show a significant change in the abundance of their transcripts. Of the 63 known genes in this group, 27 encode proteins associated with the immune response. These genes are also up-regulated by secreted, soluble factors from extracellular parasites indicating that the early response does not require parasite invasion. Later during infection, genes involved in numerous host cell processes, including glucose and mevalonate metabolism, are modulated. Many of these late genes are dependent on the direct presence of the parasite; i.e. secreted products from either the parasite or infected cells are insufficient to induce these changes. These results reveal several previously unknown effects on the host cell and lay the foundation for detailed analysis of their role in the host-pathogen interaction.

  View details for Web of Science ID 000169531100131

  View details for PubMedID 11294868

• Resistance as a tool in the study of old and new drug targets in Toxoplasma DRUG RESISTANCE UPDATES McFadden, D. C., Camps, M., Boothroyd, J. C. 2001; 4 (2): 79-84

  Abstract

  Drug resistance generated in vitro in the protozoan parasite Toxoplasma gondii is described. We focus on drugs that are in use in patients, that show some promise for such use, or that represent lead compounds for further development. No instance has yet been reported where resistance to any of these drugs has arisen in a patient or in the field although different strains do show varying degrees of sensitivity. For many of these drugs, however, resistant lines have been generated in the laboratory and these have proven very useful for elucidating a given drug's target. These targets range from metabolic pathways in the cytosol to organellar functions encoded in the mitochondrion or plastid. Such information makes predictions about how fast resistance will arise in the field but more importantly, it helps identify targets that are crucial to the parasite and predicts which combinations of drugs should act synergistically.

  View details for DOI 10.1054/drup.2001.0184

  View details for Web of Science ID 000172933600001

  View details for PubMedID 11512524

• Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene JOURNAL OF CLINICAL MICROBIOLOGY Grigg, M. E., Boothroyd, J. C. 2001; 39 (1): 398-400

  Abstract

  Sequence analysis at the 35-fold-repetitive B1 locus identified three restriction sites capable of discriminating type I (mouse-virulent) from type II or III (mouse-avirulent) strains of Toxoplasma gondii. B1 PCR-restriction fragment length polymorphism analysis of 8 type I, 17 type II, and 8 type III strains confirms the specificity of the assay. It should now be possible to ask whether strain genotype affects the severity and type of clinical disease in humans.

  View details for Web of Science ID 000166468900074

  View details for PubMedID 11136812

• Isolation and characterization of a cold-sensitive attachment/invasion mutant of Toxoplasma gondii EXPERIMENTAL PARASITOLOGY Uyetake, L., Ortega-Barria, E., Boothroyd, J. C. 2001; 97 (1): 55-59

  View details for Web of Science ID 000167201100008

  View details for PubMedID 11207115

• Success and virulence in the AIDS pathogen Toxoplasma as the result of sexual recombination between two distinct ancestries Science Grigg ME, Suzuki Y, Boothroyd JC 2001; 294: 161-16
• Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells INFECTION AND IMMUNITY Hehl, A. B., Lekutis, C., Grigg, M. E., Bradley, P. J., Dubremetz, J. F., Ortega-Barria, E., Boothroyd, J. C. 2000; 68 (12): 7078-7086

  Abstract

  Proteins with constitutive or transient localization on the surface of Apicomplexa parasites are of particular interest for their potential role in the invasion of host cells. We describe the identification and characterization of TgAMA1, the Toxoplasma gondii homolog of the Plasmodium apical membrane antigen 1 (AMA1), which has been shown to elicit a protective immune response against merozoites dependent on the correct pairing of its numerous disulfide bonds. TgAMA1 shows between 19% (Plasmodium berghei) and 26% (Plasmodium yoelii) overall identity to the different Plasmodium AMA1 homologs and has a conserved arrangement of 16 cysteine residues and a putative transmembrane domain, indicating a similar architecture. The single-copy TgAMA1 gene is interrupted by seven introns and is transcribed into an mRNA of approximately 3.3 kb. The TgAMA1 protein is produced during intracellular tachyzoite replication and initially localizes to the micronemes, as determined by immunofluorescence assay and immunoelectron microscopy. Upon release of mature tachyzoites, TgAMA1 is found distributed predominantly on the apical end of the parasite surface. A approximately 54-kDa cleavage product of the large ectodomain is continuously released into the medium by extracellular parasites. Mouse antiserum against recombinant TgAMA1 blocked invasion of new host cells by approximately 40%. This and our inability to produce a viable TgAMA1 knock-out mutant indicate that this phylogenetically conserved protein fulfills a key function in the invasion of host cells by extracellular T. gondii tachyzoites.

  View details for Web of Science ID 000167020000075

  View details for PubMedID 11083833

• Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress MOLECULAR AND CELLULAR BIOLOGY Black, M. W., Arrizabalaga, G., Boothroyd, J. C. 2000; 20 (24): 9399-9408

  Abstract

  Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. Invasion and egress by this protozoan parasite are rapid events that are dependent upon parasite motility and appear to be directed by fluctuations in intracellular [Ca(2+)]. Treatment of infected host cells with the calcium ionophore A23187 causes the parasites to undergo rapid egress in a process termed ionophore-induced egress (IIE). In contrast, when extracellular parasites are exposed to this ionophore, they quickly lose infectivity (termed ionophore-induced death [IID]). From among several Iie(-) mutants described here, two were identified that differ in several attributes, most notably in their resistance to IID. The association between the Iie(-) and Iid(-) phenotypes is supported by the observation that two-thirds of mutants selected as Iid(-) are also Iie(-). Characterization of three distinct classes of IIE and IID mutants revealed that the Iie(-) phenotype is due to a defect in a parasite-dependent activity that normally causes infected host cells to be permeabilized just prior to egress. Iie(-) parasites underwent rapid egress when infected cells were artificially permeabilized by a mild saponin treatment, confirming that this step is deficient in the Iie(-) mutants. A model is proposed that includes host cell permeabilization as a critical part of the signaling pathway leading to parasite egress. The fact that Iie(-) mutants are also defective in early stages of the lytic cycle indicates some commonality between these normal processes and IIE.

  View details for Web of Science ID 000168366100029

  View details for PubMedID 11094090

• Trans-spliced L30 ribosomal protein mRNA of Trypanosoma brucei is not subject to autogenous feedback control at the messenger RNA level MOLECULAR AND BIOCHEMICAL PARASITOLOGY Wilson, K., Uyetake, L., Boothroyd, J. C. 2000; 111 (1): 199-205

  View details for Web of Science ID 000165702100018

  View details for PubMedID 11087929

• Toxoplasma gondii: Identification of a developmentally regulated family of genes related to SAG2 EXPERIMENTAL PARASITOLOGY Lekutis, C., Ferguson, D. J., Boothroyd, J. C. 2000; 96 (2): 89-96

  Abstract

  Previous studies have shown the surface of Toxoplasma gondii to be dominated by a family of proteins closely related to SAG1. In this study, we report the existence of a second family of genes defined by homology to SAG2. The predicted amino acid sequences of these three new proteins suggests that they are all glycosylphosphatidylinositol-linked surface antigens. All three also contain N-linked glycosylation sites, although their use has yet to be demonstrated. One of these SAG2-related antigens, SAG2B, is expressed in tachyzoites with an apparent size of 23 kDa. It is distinct, however, from the previously identified P23. In contrast to SAG2B, SAG2C and SAG2D appear to be expressed exclusively on the surface of bradyzoites. Analysis of the SAG2 family shows it to have weak but significant homology to the SAG1 family. Thus, all of the sequenced surface antigens of tachyzoites and many of those of bradyzoites fall into one large superfamily that can be divided into two subgroups defined by the prototypic and highly immunogenic SAG1 and SAG2, respectively.

  View details for Web of Science ID 000165137000004

  View details for PubMedID 11052867

• Lytic cycle of Toxoplasma gondii MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS Black, M. W., Boothroyd, J. C. 2000; 64 (3): 607-?

  Abstract

  Toxoplasma gondii is an obligate intracellular pathogen within the phylum Apicomplexa. This protozoan parasite is one of the most widespread, with a broad host range including many birds and mammals and a geographic range that is nearly worldwide. While infection of healthy adults is usually relatively mild, serious disease can result in utero or when the host is immunocompromised. This sophisticated eukaryote has many specialized features that make it well suited to its intracellular lifestyle. In this review, we describe the current knowledge of how the asexual tachyzoite stage of Toxoplasma attaches to, invades, replicates in, and exits the host cell. Since this process is closely analogous to the way in which viruses reproduce, we refer to it as the Toxoplasma "lytic cycle."

  View details for Web of Science ID 000089249600007

  View details for PubMedID 10974128

• Characterization of cytochrome b from Toxoplasma gondii and Q(o) domain mutations as a mechanism of atovaquone-resistance MOLECULAR AND BIOCHEMICAL PARASITOLOGY McFadden, D. C., Tomavo, S., Berry, E. A., Boothroyd, J. C. 2000; 108 (1): 1-12

  Abstract

  Atovaquone is active in vitro against the tachyzoites of Toxoplasma gondii at nanomolar concentrations and is used clinically to treat acute cases of human toxoplasmosis. In pursuit of the mechanism of action of atovaquone against T. gondii and to understand how resistance might arise, drug-resistant mutants were generated and examined. The previously uncloned cytochrome b gene of T. gondii was cloned and sequenced from wild type and resistant strains as this was a likely candidate for the target of the drug and thus a source of resistance. Mutations are present within the cytochrome b gene of atovaquone-resistant parasites (M129L and I254L) and represent alterations in two different regions of the ubiquinol-binding pocket (Q(o) domain) of cytochrome b, suggesting that atovaquone interferes with electron transport at the cytochrome bc(1) complex in T. gondii. A structural model for how this hydroxynaphthoquinone is binding within the Q(o) domain is presented. Further analysis of the cytochrome b gene suggested that the protein may differ from other homologues by terminating within the mitochondrial membrane. Cytochrome b becomes the first complete mitochondrial gene and cognate protein to be described for T. gondii.

  View details for Web of Science ID 000087166900001

  View details for PubMedID 10802314

• Cytochrome b mutation identified in a decoquinate-resistant mutant of Toxoplasma gondii 5th International Workshop on Opportunistic Protists McFadden, D. C., Boothroyd, J. C. WILEY-BLACKWELL PUBLISHING, INC. 1999: 81S–82S

  View details for Web of Science ID 000082958900052

  View details for PubMedID 10519259

• Identification of the pro-mature processing site of Toxoplasma ROP1 by mass spectrometry MOLECULAR AND BIOCHEMICAL PARASITOLOGY Bradley, P. J., Boothroyd, J. C. 1999; 100 (1): 103-109

  Abstract

  The rhoptries are specialized secretory organelles that function during host cell invasion in the obligate intracellular parasite Toxoplasma gondii. All T. gondii rhoptry proteins studied to date are synthesized as pro-proteins that are then processed to their mature forms. To understand the role of the pro region in rhoptry protein function, we have precisely defined the processing site of the pro-region of the rhoptry protein ROP1. Efforts to determine such processing sites have been prevented by blocked N-termini of mature proteins isolated from T. gondii. To overcome this problem, we have used an engineered form of ROP1 and mass spectrometry to demonstrate that proROP1 is processed to its mature form between the glutamic acid at position 83 and alanine at position 84. These data also show that mature ROP1 lacks substantial post-translational modifications, a result which has important implications for targeting of rhoptry proteins.

  View details for Web of Science ID 000080576600011

  View details for PubMedID 10376998

• Trypanosoma brucei: Cis-acting sequences involved in the developmental regulation of PARP expression EXPERIMENTAL PARASITOLOGY Wilson, K., Uyetake, L., Boothroyd, J. 1999; 91 (3): 222-230

  Abstract

  The procyclic acidic repetitive protein (PARP or procyclin) of the parasitic protozoan Trypanosoma brucei is a developmentally regulated protein that shows extreme differences in its level of expression in different stages of the parasite's life cycle. Specifically, it is the major surface protein in the procyclic (insect) stage and, although the PARP gene is being actively transcribed in the mammalian bloodstream stage, there is no detectable PARP mRNA or protein in these cells. The 3'-untranslated region (UTR) of PARP, as well as other trypanosome genes, has the ability to confer the appropriate developmental regulation pattern onto chimeric reporter genes. To understand the mechanism of posttranscriptional regulation, selective replacement mutagenesis of the PARP mRNA 3'UTR was done to identify the cis-acting sequences involved in the down-regulation of this mRNA in bloodstream-form T. brucei. Transient transformation of constructs containing the PARP promoter and 5'UTR, the beta-glucuronidase coding region, and the selectively mutagenized or unaltered PARP 3'UTR were performed in procyclic and bloodstream T. brucei. The results of the reporter gene assays on the transformed cells indicate that there are at least two elements in the PARP 3'UTR which in bloodstream cells are involved in regulation of PARP expression and which appear to function as negative elements. In procyclic cells, there are two regions in which mutagenesis indicates positive cis-regulatory sequences, one of which has been previously defined (A. Hehl et al., 1994, Proc. Natl. Acad. Sci. USA 91, 370-374). These results indicate that multiple cis-acting elements within the PARP 3'UTR are involved in the developmental regulation of PARP expression and that regulation is controlled in a complex manner, presumably involving several cellular trans-acting factors.

  View details for Web of Science ID 000079317400003

  View details for PubMedID 10072324

• A Toxoplasma lectin-like activity specific for sulfated polysaccharides is involved in host cell infection JOURNAL OF BIOLOGICAL CHEMISTRY Ortega-Barria, E., Boothroyd, J. C. 1999; 274 (3): 1267-1276

  Abstract

  Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

  View details for Web of Science ID 000079956900015

  View details for PubMedID 9880495

• Molecular biology's lessons about Toxoplasma development: Stage-specific homologs PARASITOLOGY TODAY Knoll, L. J., Boothroyd, J. C. 1998; 14 (12): 490-493

  Abstract

  Within intermediate hosts (such as humans), the protozoan parasite Toxoplasma gondii has two life cycle stages: a rapidly replicating form called a tachyzoite and a slowly growing, quiescent form called a bradyzoite. Recently, molecular biology studies have shown that tachyzoites and bradyzoites express a number of homologs (ie. evolutionary related genes)expressed exclusively in one or the other stage. Here, Laura Knoll and John Boothroyd describe examples of how these stage-specific homologs were discovered, and speculate about their regulation and functional significance.

  View details for Web of Science ID 000077411100007

  View details for PubMedID 17040862

• Identification of a nuclear protein in Trypanosoma brucei with homology to RNA-binding proteins from cis-splicing systems MOLECULAR AND BIOCHEMICAL PARASITOLOGY Manger, I. D., Boothroyd, J. C. 1998; 97 (1-2): 1-11

  Abstract

  Gene expression in trypanosomes is controlled at the level of pre-mRNA maturation via trans-splicing and polyadenylation and through changes in mRNA stability. To identify the trans- acting factors involved in this regulation, we have used a degenerate PCR approach to clone genes encoding the RNA recognition motif (RRM) consensus. We have identified a single-copy gene encoding a protein (designated RRM1) which contains three consensus RRM motifs, two tandem copies of a retroviral gag-like CCHC 'zinc finger' and an arginine-serine (RS) rich region. Western blotting indicates that RRM1 is expressed in both procyclic and bloodstream-form trypanosomes and has an apparent mobility on SDS-PAGE of ca. 70 Kd. RRM1 is localized in the trypanosome nucleus in substructures which may be functionally analogous to the 'speckles' associated with cis-splicing in higher eukaryotic cells. The structure of RRM1, its pattern of expression and its intracellular location suggest that it may play a role in trans-splicing.

  View details for Web of Science ID 000077776100001

  View details for PubMedID 9879882

• Processing of Toxoplasma ROP1 protein in nascent rhoptries MOLECULAR AND BIOCHEMICAL PARASITOLOGY Soldati, D., Lassen, A., Dubremetz, J. F., Boothroyd, J. C. 1998; 96 (1-2): 37-48

  Abstract

  Secretion in the obligate intracellular parasite, Toxoplasma gondii, occurs through a number of regulated compartments. Among these are the apical organelles known as rhoptries which release their contents as part of the invasion process. We are interested in the processing, targeting and ultimate function of rhoptry proteins (and have focused our analyses on rhoptry protein 1 (ROP1). In this paper, we address the issue of processing: using a number of engineered forms of the ROP1 gene (introduced into a ROP1- background), we show that ROP1 is synthesized as a pre-pro-protein that is subject to proteolytic cleavages to remove the pre-sequence and the 'pro' region, at the N-terminus. Using brefeldin A (BFA) and reduced temperature we show that this processing occurs late in the secretory pathway of the parasite. Immunolocalization studies with epitope-tagged constructs indicate that processing is apparently occurring in the nascent rhoptries of dividing parasites. The results are discussed in the context of the targeting and possible function of the ROP1 protein.

  View details for Web of Science ID 000077240200004

  View details for PubMedID 9851605

• The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1 INFECTION AND IMMUNITY Manger, I. D., Hehl, A. B., Boothroyd, J. C. 1998; 66 (5): 2237-2244

  Abstract

  Toxoplasma gondii is an Apicomplexan parasite with a complex life cycle that includes a rapidly dividing asexual stage known as the tachyzoite. The tachyzoite surface has been reported to comprise five major antigens, the most abundant of which is designated SAG1 (for surface antigen 1). At least one of the other four (SAG3) and another recently described minor antigen (SRS1 [for SAG1-related sequence 1]) have previously been shown to be structurally related to SAG1. To determine if further SAG1 homologs exist, we searched a Toxoplasma expressed sequence tag (EST) database and found numerous ESTs corresponding to at least three new genes related to SAG1. Like SAG1, these new SRS genes encode apparently glycosylphosphatidylinositol-anchored proteins that share several motifs and a set of conserved cysteine residues. This family appears to have arisen by divergence from a common ancestor under selection for the conservation of overall topology. The products of two of these new genes (SRS2 and SRS3) are shown to be expressed on the surface of Toxoplasma tachyzoites by immunofluorescence. We also identified strain-specific differences in relative expression levels. A total of 10 members of the SAG1 gene family have now been identified, which apparently include three of the five major surface antigens previously described and one antigen expressed only in bradyzoites. The function of this family may be to provide a redundant system of receptors for interaction with host cells and/or to direct the immune responses that limit acute T. gondii infections.

  View details for PubMedID 9573113

• Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: Identification of developmentally regulated genes INFECTION AND IMMUNITY Manger, I. D., Hehl, A., Parmley, S., Sibley, L. D., Marra, M., Hillier, L., Waterston, R., Boothroyd, J. C. 1998; 66 (4): 1632-1637

  Abstract

  Toxoplasma gondii is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on approximately 7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18-28, 1998). Here, we report the results from analysis of approximately 2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in Toxoplasma.

  View details for Web of Science ID 000072706100050

  View details for PubMedID 9529091

• Development of a stable episomal shuttle vector for Toxoplasma gondii JOURNAL OF BIOLOGICAL CHEMISTRY Black, M. W., Boothroyd, J. C. 1998; 273 (7): 3972-3979

  Abstract

  The rapid developments in the molecular genetics of Toxoplasma gondii have far reaching implications in treatment and vaccination strategies for this as well as closely related pathogens such as Plasmodium. Although stable transformation of this parasite through homologous and illegitimate genomic integration has provided many of the tools necessary for genetic analysis, subsequent manipulations of the DNA have proven laborious. This report describes the selection and subsequent characterization of a Toxoplasma sequence that permits the episomal maintenance of bacterial plasmids in this parasite. This sequence was isolated from the Toxoplasma genome through selection for episomal stability of a pUC19-based library in the absence of a selectable marker. A 500-base pair fragment was determined to possess the stabilization activity. Transformations of Toxoplasma using vectors possessing this fragment, referred to as EMS (episomal maintenance sequence), demonstrated an elevated stable transformation frequency compared with the vector alone. Mutants deficient in hypoxanthine-xanthine-guanine phosphoribosyltransferase activity were used as a test to see if this gene could be selected from a genomic library using a vector containing the EMS. The success of this test demonstrates the utility of EMS-containing vectors in complementation strategies and the ability of such constructs bearing large fragments of the Toxoplasma genome to be maintained episomally.

  View details for Web of Science ID 000072048400027

  View details for PubMedID 9461585

• Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase MOLECULAR AND CELLULAR BIOLOGY Knoll, L. J., Boothroyd, J. C. 1998; 18 (2): 807-814

  Abstract

  Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

  View details for Web of Science ID 000071716000016

  View details for PubMedID 9447977

• The surface of Toxoplasma: More and less 10th International Congress of Protozoology Boothroyd, J. C., Hehl, A., Knoll, L. J., Manger, I. D. ELSEVIER SCI LTD. 1998: 3–9

  Abstract

  As for any intracellular parasite, the surface of the Apicomplexan parasite Toxoplasma gondii must fulfil many functions including a role in attachment, signalling, invasion, transport and interaction with the immune response of the host. In this review, we describe the current state of knowledge on the molecules that are found on the surface of the different developmental stages of this parasite and speculate as to how at least some of these multiple functions are fulfilled. Special emphasis is given to the growing family of surface antigens that are related to the tachyzoite-specific surface antigen 1. We conclude that the surface (of tachyzoites, at least) is both more and less complex than previously thought: there are more proteins present but their sequences suggest that the majority may share a similar overall structure typified by surface antigen 1.

  View details for Web of Science ID 000072005200002

  View details for PubMedID 9504330

• Analysis of Toxoplasma gondii stably transfected with a transmembrane variant of its major surface protein, SAG1 JOURNAL OF CELL SCIENCE Seeber, F., Dubremetz, J. F., Boothroyd, J. C. 1998; 111: 23-29

  Abstract

  We have genetically engineered Toxoplasma gondii so that its major surface antigen SAG1 is anchored by a human transmembrane domain (SAG1-TM) instead of its natural GPI anchor (SAG1-GPI) in order to initiate studies to address the function of this protein anchor in parasitic protozoa as well as to get insights into the functional role of SAG1. Our results show that SAG1-TM is correctly folded (at least as judged by the presence of conformationally dependent epitopes) and targeted to the surface of the parasite, indicating that the GPI anchor does not determine its localization nor overall three-dimensional structure. No significant difference was seen in any aspect of the growth of the SAG1-TM mutant. However, compared to the natural SAG1-GPI, SAG1-TM does not form strong associations with itself and/or other molecules in high molecular weight complexes suggesting that allowing such complexes to form may be one role of the GPI anchor. The in vitro half-life of SAG1-TM of extracellular parasites is significantly lower than that of SAG1-GPI suggesting a stabilizing function of the glycolipid anchor against degradation and/or membrane release. Antibodies to SAG1 are shed from SAG1-TM parasites as they invade, just as they are stripped from SAG1-GPI bearing parasites. The stripping, therefore, is unlikely to be driven by the action of lipases.

  View details for Web of Science ID 000071667000003

  View details for PubMedID 9394009

• Gene discovery by EST sequencing in Toxoplasma gondii reveals sequences restricted to the apicomplexa GENOME RESEARCH Ajioka, J. W., Boothroyd, J. C., Brunk, B. P., Hehl, A., Hillier, L., Manger, I. D., Marra, M., Overton, G. C., Roos, D. S., Wan, K. L., Waterston, R., Sibley, L. D. 1998; 8 (1): 18-28

  Abstract

  To accelerate gene discovery and facilitate genetic mapping in the protozoan parasite Toxoplasma gondii, we have generated >7000 new ESTs from the 5' ends of randomly selected tachyzoite cDNAs. Comparison of the ESTs with the existing gene databases identified possible functions for more than 500 new T. gondii genes by virtue of sequence motifs shared with conserved protein families, including factors involved in transcription, translation, protein secretion, signal transduction, cytoskeleton organization, and metabolism. Despite this success in identifying new genes, more than 50% of the ESTs correspond to genes of unknown function, reflecting the divergent evolutionary status of this parasite. A newly recognized class of genes was identified based on its similarity to sequences known only from other members of the same phylum, therefore identifying sequences that are apparently restricted to the Apicomplexa. Such genes may underlie pathways common to this group of medically important parasites, therefore identifying potential targets for intervention.

  View details for Web of Science ID 000071807000004

  View details for PubMedID 9445484

• Identification and characterization of SRS1, a Toxoplasma gondii surface antigen upstream of and related to SAG1 MOLECULAR AND BIOCHEMICAL PARASITOLOGY Hehl, A., Krieger, T., Boothroyd, J. C. 1997; 89 (2): 271-282

  Abstract

  Previous investigations of the major surface antigen (SAG1) promoter of Toxoplasma gondii indicated an ability to function bi-directionally in transient transformation assays at least. This suggests there might be another tachyzoite-specific gene being divergently transcribed from the SAG1 promoter in its normal chromosomal location. To investigate this possibility we have characterized the region upstream of SAG1 and report here a co-directional transcription unit coding for a probable GPI-anchored surface protein with homology to SAG1 and SAG3. This antigen, which had not previously been identified in surface iodination experiments is given the acronym SRS1, for SAG1-related sequence 1. Genomic organization and sequence of a full-length cDNA of SRS1 are presented. Antisera against a recombinant SRS1 protein produced in Escherichia coli, recognize a specific band of 46 kDa in parasite lysates which corresponds to the largest of the GPI-anchored proteins by Western blot. The possible role of this previously unidentified surface antigen is discussed.

  View details for Web of Science ID A1997YB61600010

  View details for PubMedID 9364971

• Genetic analysis in toxoplasma: Gene discovery with expressed sequence tags and rapid mapping of natural polymorphisms METHODS Hehl, A., Manger, I. D., Boothroyd, J. C. 1997; 13 (2): 89-102

  Abstract

  Genetic analysis of the protozoan parasite Toxoplasma gondii has undergone a rapid expansion in recent years. This is due to effort in a number of laboratories that have worked on the development of molecular genetic techniques. It is also due, however, to the natural biology of this system (including a well-described sexual cycle) that makes possible genetic mapping of the F1 progeny from a cross. In this article, we present a detailed methodology for rapidly mapping natural polymorphisms between the ME49 and CEP strains for which extensive restriction fragment length polymorphism analysis has already been performed. The example we present shows that the failure to detect expression of bradyzoite-specific surface antigens in the CEP strain under conditions that promote differentiation in vitro is not a result of a general failure to express such genes; instead, it is apparently due to antigenic polymorphism in the gene products concerned. This conclusion was reached rapidly and definitively by genetic mapping, whereas molecular approaches would have taken considerably longer. We also show how the recent effort to create an extensive database of expressed sequence tags for this parasite can promote the very rapid discovery of genes that reveal much about the biology of Toxoplasma. The example presented deals with the expression of a family of closely related surface antigens in the tachyzoite stage.

  View details for Web of Science ID A1997YL34200003

  View details for PubMedID 9405193

• Genetic and biochemical analysis of development in Toxoplasma gondii Discussion Meeting of the Royal-Society-of-London on Immune Effector Mechanisms in Parasitic Infections Boothroyd, J. C., Black, M., Bonnefoy, S., Hehl, A., Knoll, L. J., Manger, I. D., ORTEGABARRIA, E., Tomavo, S. ROYAL SOC. 1997: 1347–54

  Abstract

  Toxoplasma gondii has recently come under intense study as a model for intracellular parasitism because it has a number of properties that facilitate experimental manipulation. Attention is now being turned towards understanding the developmental biology of this complex parasite. The differentiation between the two asexual stages, the rapidly growing tachyzoites and the more slowly dividing, encysted bradyzoites, is of particular interest. Progression from the former to the latter is influenced by the host's immune response. This paper describes current progress on a number of research fronts, all aimed at understanding the triggers that push the tachyzoite-bradyzoite equilibrium in one or other direction and the changes that occur in gene expression (and ultimately metabolism and function). Chief among the techniques used for these studies are genetics and molecular genetics. Recent progress in these areas is described.

  View details for Web of Science ID A1997YA54400008

  View details for PubMedID 9355126

• Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro ANTIMICROBIAL AGENTS AND CHEMOTHERAPY McFadden, D. C., Seeber, F., Boothroyd, J. C. 1997; 41 (9): 1849-1853

  Abstract

  A microtiter assay for drug evaluation has been developed with a strain of Toxoplasma gondii that expresses bacterial beta-galactosidase. By using chlorophenol red-beta-D-galactopyranoside (CPRG) as the substrate for beta-galactosidase, the efficacy of a drug against the parasite can be determined with a colorimetric readout. Drugs known to have activity against T. gondii (specifically, pyrimethamine, sulfadiazine, atovaquone, and clindamycin) were tested, and efficacies were determined by CPRG cleavage. The 50% inhibitory concentrations determined by the CPRG-based colorimetric assay were similar to those determined by the traditional radiolabelled uracil incorporation assay. Since CPRG is nontoxic to the parasite, viable drug-treated parasites can be obtained at the conclusion of the assay for further evaluation if desired. This assay provides a high-throughput and nonradioactive alternative for the identification of anti-T. gondii compounds.

  View details for Web of Science ID A1997XV70100001

  View details for PubMedID 9303372

• Implications of conserved structural motifs in disparate trypanosome surface proteins. MOLECULAR AND BIOCHEMICAL PARASITOLOGY Carrington, M., Boothroyd, J. 1996; 81 (2): 119-126

  Abstract

  Evasion of the host immune system by Trypanosoma brucei is dependent on the sequential expression of individual genes encoding antigenically distinct variant surface glycoproteins (VSG). VSGs are antigenically distinct due to extensive differences in primary sequence; the only obvious conserved feature in the primary sequence is the location of cysteines that form disulphide bridges. Despite this difference, it is believed that VSGs have a conserved tertiary structure which could explain how a range of VSGs with different primary sequences can perform the same apparent function of producing a monolayer barrier that prevents the host antibodies from recognising other cell surface proteins. The main feature of the VSG tertiary structure is two long alpha-helices per monomer that are perpendicular to the cell surface and define the elongated shape of the VSG. The alpha-helices can be identified in the primary sequence by heptad analysis. Here, we briefly review the current understanding of VSG structure and discuss the fact that the cysteine residues and the heptads are conserved in some non-VSG surface proteins from T. brucei, providing strong evidence that these share a similar tertiary structure. These findings suggest that this master structure has evolved to facilitate a range of functions and has implications for understanding the architecture of the trypanosome cell surface and the origins of antigenic variation.

  View details for Web of Science ID A1996VM86500001

  View details for PubMedID 8898328

• Sequence divergence in a family of variant surface glycoprotein genes from trypanosomes: Coding region hypervariability and downstream recombinogenic repeats JOURNAL OF MOLECULAR EVOLUTION Field, M. C., Boothroyd, J. C. 1996; 42 (5): 500-511

  Abstract

  The surface of the parasitic protozoan Trypanosoma brucei spp. is covered with a dense coat consisting of a single type of glycoprotein molecule, the variant surface glycoprotein (VSG). There may be as many as 1,000 genes for VSG within the genome of T. brucei, and the switch of expression from one to another is the phenomenon of antigenic variation. As an approach to understanding the evolution of VSG genes we have determined the genomic DNA sequences of the eight genes encoding the variant surface glycoprotein 117 (VSG) family. From these data we have observed a number of features concerning the relationships between these genes: (1) there is a region of high variability confined to the N-terminus of the coding sequence, and comparison of the sequences with the available X-ray diffraction crystal structures suggests that two of the most variable stretches within the N-terminal domain are present on surface-exposed loops, indicating a role for epitope selection in evolution of these genes; (2) the 29 nucleotides surrounding the splice acceptor site are absolutely conserved in all eight 117 VSG genes; (3) numerous insertion/deletion mutations are located within or immediately downstream of the C-terminal protein-coding sequences: (4) within 500 bp downstream of the insertion/deletion mutations are one or two copies of a repeat motif highly homologous to the recombinogenic 76-bp repeat sequences present upstream of many VSG basic copy genes and the expression-linked copy.

  View details for Web of Science ID A1996UR42300004

  View details for PubMedID 8662002

• Escherichia coli beta-galactosidase as an in vitro and in vivo reporter enzyme and stable transfection marker in the intracellular protozoan parasite Toxoplasma gondii GENE Seeber, F., Boothroyd, J. C. 1996; 169 (1): 39-45

  Abstract

  We have developed several protocols for the use of beta-galactosidase (betaGal) from Escherichia coli as a reporter enzyme in transfection studies of Toxoplasma gondii (Tg) and as a readily screenable marker for stable transformation. Three Tg expression vectors with different promoters driving lacZ were constructed and shown in transient transfections to differ in their relative expression levels. Using a fluorescent betaGal substrate, it was possible to detect enzymatic activity with as little as 50 ng of transfected lacZ-containing plasmid DNA. When stably transformed intracellular parasites were cultivated in microtiter plates in the presence of the color substrate, chorophenol red-beta-D-galactopyranoside (CPRG), the signal from as few as 400 Tg could be readily detected by eye. Using serial dilutions of transfected parasite cultures in the presence of CPRG, we were able to clone stably expressing betaGal-positive Tg without the need for another selectable marker. Such lacZ transgenics could also be visualized histochemically in the tissue of infected mice. Thus, the application of betaGal to studies on Tg provides not only a much needed second reporter for transient transfection, it also comprises a safe and sensitive marker for the generation and analysis of stably transfected parasites.

  View details for Web of Science ID A1996TX64000006

  View details for PubMedID 8635747

• Use of chimeric recombinant polypeptides to analyse conformational, surface epitopes on trypanosome variant surface glycoproteins MOLECULAR MICROBIOLOGY Hsia, R. C., Beals, T., Boothroyd, J. C. 1996; 19 (1): 53-63

  Abstract

  Identification of surface-exposed epitopes on the variant surface glycoproteins (VSGs) of African trypanosomes has been complicated by the observation that most such epitopes are highly conformational. As a result, whenever the molecule is broken down for analysis, the epitope is generally lost. We have exploited the existence of closely related gene families to create chimeric molecules in which particular segments of one VSG are placed in the analogous position of a related but antigenically distinct VSG. The process is used in both a positive and negative manner, so that the epitope can be specifically added or destroyed in a given chimera. As an example, we have used this approach to identify the regions involved in reactivity to a monoclonal antibody specific for VSG117 on the surface of live trypanosomes. We show that while deletion of almost any region of VSG117 results in loss of reactivity to this monoclonal antibody, substituting particular regions with the corresponding segment of the structurally related but antigenically distinct VSG FM8.5 restores reactivity in most but not all cases, thereby delimiting the antigenically key regions. Likewise, substituting key regions from VSG117 into FM8.5 confers reactivity on the resulting chimeras. This approach circumvents some of the problems that result from the highly conformational nature of VSG and should allow further elucidation of the biologically relevant antigenic topology of VSGs.

  View details for Web of Science ID A1996TQ60000006

  View details for PubMedID 8821936

• INTERCONNECTION BETWEEN ORGANELLAR FUNCTIONS, DEVELOPMENT AND DRUG-RESISTANCE IN THE PROTOZOAN PARASITE, TOXOPLASMA-GONDII 1994 Annual Meeting of the Australian-Society-for-Parasitology Tomavo, S., Boothroyd, J. C. PERGAMON-ELSEVIER SCIENCE LTD. 1995: 1293–99

  Abstract

  The protozoan parasite Toxoplasma gondii causes severe disease in animals and humans. In AIDS patients, for example, the encephalitis it produces is a major cause of death. Part of the very successful strategy adopted by the parasite centers on its ability to differentiate from the actively growing tachyzoite form to a chronic, almost latent state called the bradyzoite. The molecular signals and precise triggers involved in this differentiation process are not known. Drugs for treating toxoplasmosis are not capable of clearing the infection apparently because of their inability to eradicate the bradyzoites. Recently, as part of our efforts to understand the mode of action of a promising new drug, atovaquone, we have generated and analysed a mutant that is resistant to this drug. Surprisingly, we found that this mutant is predisposed to spontaneously differentiate from the tachyzoite to bradyzoite form in vitro (Tomavo & Boothroyd, submitted). Given that atovaquone is believed to act on the parasite mitochondria, we were interested to explore the relationship between mitochondrial function and differentiation. We find that atovaquone and a number of other drugs targeted to mitochondria will cause wild type parasites to differentiate from tachyzoites to bradyzoites suggesting some sort of adaptive response to a decrease in mitochondrial activities. The fact that atovaquone-resistant mutants are hypersensitive to clindamycin, a drug believed to work on the putative plastid of these parasites, suggests a model for how the mitochondrion and plastid interact and how they may be tied into the process and state of differentiation. This model is presented and discussed.

  View details for Web of Science ID A1995TG99000005

  View details for PubMedID 8635881

• Trypanosoma brucei: Molecular cloning of homologues of small GTP-binding proteins involved in vesicle trafficking EXPERIMENTAL PARASITOLOGY Field, M. C., Boothroyd, J. C. 1995; 81 (3): 313-320

  Abstract

  Members of the rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes. Individual rab proteins have a remarkable degree of specific subcellular localization. As a first approach to the study of these proteins in the protozoan parasite Trypanosoma brucei we have undertaken a survey of the rab subfamily using a strategy based on degenerate polymerase chain reaction utilizing the miniexon and the highly conserved WDTAGQE box which comprises part of the GTP-binding site. A number of T. brucei partial cDNAs were obtained from procyclic stage cDNA, and seven of these clones (designated rtb 1, 3-7, 9) were clearly members of the rab family. Northern analysis of poly(A)-selected RNA indicates that rtb1, 3, 4, and 7 are constitutively expressed at low levels in both life-stages of T. brucei. By Southern analysis of trypanosome genomic DNA and specific PCR from an isolated genomic clone, we show that two of the genes, RTB1 and RTB7, are adjacent or at least closely linked in the T. brucei genome, while the other five are dispersed. These data provide important molecular reagents for dissecting the unusual secretory pathway in this organism.

  View details for Web of Science ID A1995TK43100010

  View details for PubMedID 7498428

• Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Taxoplasma gondii MOLECULAR AND BIOCHEMICAL PARASITOLOGY Black, M., Seeber, F., Soldati, D., Kim, K., Boothroyd, J. C. 1995; 74 (1): 55-63

  Abstract

  This report describes the use of restriction enzyme-mediated integration (REMI) to increase the transformation frequency and allow co-transfection of several unselected constructs under the selection of a single selectable marker. We found that while BamHI (the enzyme used to originally demonstrate REMI (Schiestl, R.H. and Petes, T.D. (1991) Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Nati. Acad. Sci. USA 88, 7585-7589) increased the number of transformants by 2-5-fold over the control without added enzyme, NotI proved to be a further 29-46-times more effective in enhancing stable transformation. This simple technique was used in the transformation of three non-selective markers (two modified membrane proteins and beta-galactosidase) with a selectable construct expressing chloramphenicol acetyltransferase. Following chloramphenicol selection, four out of ten independent transformants stably acquired all four constructs with at least two expressing all four genes at the protein level. These results demonstrate that REMI may be used in the efficient stable transformation and co-transfection of this and perhaps other protozoan parasites.

  View details for Web of Science ID A1995TP84900006

  View details for PubMedID 8719245

• Complementation of a Toxoplasma gondii ROP1 knock-out mutant using phleomycin selection MOLECULAR AND BIOCHEMICAL PARASITOLOGY Soldati, D., Kim, K., Kampmeier, J., Dubremetz, J. F., Boothroyd, J. C. 1995; 74 (1): 87-97

  Abstract

  The ROP1 gene of Toxoplasma gondii encodes a rhoptry protein that has been implicated in host cell invasion by this obligate intracellular protozoan. To further explore the function of this protein, we created a ROP1 deletion mutant by transfection with a plasmid encoding the bacterial chloramphenicol acetyltransferase (cat) gene flanked by ROP1 genomic sequences. Selection for chloramphenicol resistance yielded the desired ROP1-deleted or 'knock-out' mutant. Analysis of this mutant both in vitro and in vivo shows no significant alterations in growth rate, host specificity, invasiveness or virulence and thus the ROP1 gene is not obligatory for the RH strain, at least under the conditions tested. However, electron microscopy reveals that the mutant strain's rhoptries are altered in ultrastructure; they are thinner and homogeneously electron-dense compared with the thicker and normally mottled or honeycombed appearance of wild-type rhoptries. The knock-out mutant was rescued using co-transfection of a cosmid carrying the ROP1 gene together with a plasmid encoding a new selectable marker for T. gondii, the bleomycin resistance gene (ble) from Streptoalloteichus. Southern blot analysis showed that both DNAs were stably integrated into the Toxoplasma genome, although not into the ROPI locus. The resulting strain showed wild-type levels of ROP1 expression and rescue of the ultrastructural phenotype (i.e., the rhoptries returned to their normal, mottled appearance), thus establishing a cause/effect relationship between the absence of ROP1 and the electron-opacity. These results demonstrate the utility of the reverse genetic approach in the study of Toxoplasma gene function and provide a further selectable marker for such manipulations.

  View details for Web of Science ID A1995TP84900009

  View details for PubMedID 8719248

• TOXOPLASMA-GONDII - STABLE COMPLEMENTATION OF SAG1 (P-30) MUTANTS USING SAG1 TRANSFECTION AND FLUORESCENCE-ACTIVATED CELL SORTING EXPERIMENTAL PARASITOLOGY Kim, K., Boothroyd, J. C. 1995; 80 (1): 46-53

  Abstract

  Toxoplasma gondii and the related Apicomplexan protozoan pathogens, Plasmodium, Cryptosporidium, and Eimeria, are obligate intracellular parasites which cause severe disease in their hosts. The recent development of transient transfection of Toxoplasma permits the development of strategies utilizing "reverse genetics" to identify molecules critical to parasite survival within the host. We have utilized transfection of Toxoplasma tachyzoites to stably complement a sag1 (or p30) mutant that does not make detectable SAG1. Transfection of mutants with the wild-type SAG1 gene resulted in transient expression of SAG1 in approximately 15-20% of the transfected population. Stable transformants were enriched by repeated sorting of live parasites using a fluorescein-labeled monoclonal antibody specific for SAG1. Cloned recombinant parasites expressed SAG1 at wild-type levels and maintained expression for over 5 months after transfection (approximately 300 divisions). Cloned transformants (which proved to be siblings) carried both the mutated gene and one copy of the transfected gene which had inserted randomly into the Toxoplasma genome.

  View details for Web of Science ID A1995QD58900006

  View details for PubMedID 7821410

• A HOMOLOG OF THE NUCLEAR GTPASE RAN/TC4 FROM TRYPANOSOMA-BRUCEI MOLECULAR AND BIOCHEMICAL PARASITOLOGY Field, M. C., Field, H., Boothroyd, J. C. 1995; 69 (1): 131-134

  View details for Web of Science ID A1995QE15000017

  View details for PubMedID 7723783

• A SELECTOR OF TRANSCRIPTION INITIATION IN THE PROTOZOAN PARASITE TOXOPLASMA-GONDII MOLECULAR AND CELLULAR BIOLOGY Soldati, D., Boothroyd, J. C. 1995; 15 (1): 87-93

  Abstract

  The recent development of an efficient transfection system for the apicomplexan Toxoplasma gondii allows a comprehensive dissection of the elements involved in gene transcription in this obligate intracellular parasite. We demonstrate here that for the SAG1 gene, a stretch of six repeated sequences in the region 35 to 190 bp upstream of the first of two transcription start sites is essential for efficient and accurate transcription initiation. This repeat element shows characteristics of a selector in determining the position of the transcription start sites.

  View details for Web of Science ID A1995PX95300011

  View details for PubMedID 7799972

• Toxoplasma workshop overview. journal of eukaryotic microbiology Schwartzman, J. D., Boothroyd, J. C., Kasper, L. H. 1994; 41 (5): 19S-21S

  View details for PubMedID 7804224

• CONFORMATIONALLY APPROPRIATE EXPRESSION OF THE TOXOPLASMA ANTIGEN SAG1 (P-30) IN CHO CELLS INFECTION AND IMMUNITY Kim, K., Bulow, R., Kampmeier, J., Boothroyd, J. C. 1994; 62 (1): 203-209

  Abstract

  The Toxoplasma gondii major surface antigen, called SAG1 or p30, is a highly immunogenic protein which has generated great interest as a diagnostic reagent, as a potential subunit vaccine, and for its role in invasion. Unfortunately, bacterial recombinant protein is grossly misfolded so that, for example, it is not effectively recognized by antibodies to native SAG1. To overcome this, we have turned to expression in CHO cells, using cotransfection of the SAG1 gene and the mouse dihydrofolate reductase (DHFR) gene into CHO cells that are DHFR-. SAG1 expression was amplified by methotrexate coselection of CHO cells in combination with fluorescence-activated cell sorting for SAG1 expression. The resulting population expressed recombinant SAG1 that is recognized by antiserum specific for natural, nonreduced SAG1, indicating that, unlike in bacteria, expression in CHO cells results in proper folding. Processing was at least partially correct in that, like natural SAG1, recombinant SAG1 was attached to the plasma membrane via a glycolipid anchor, although tunicamycin treatment was necessary to prevent N-glycosylation (SAG1 is not glycosylated in the parasite but does have a consensus N-linked site). Finally, purified recombinant SAG1 was recognized by human sera known to be reactive to toxoplasma proteins, indicating that this material has potential as a diagnostic reagent and possibly as a component of a subunit vaccine.

  View details for Web of Science ID A1994MN48800029

  View details for PubMedID 8262628

• GENE REPLACEMENT IN TOXOPLASMA-GONDII WITH CHLORAMPHENICOL ACETYLTRANSFERASE AS SELECTABLE MARKER SCIENCE Kim, K., Soldati, D., Boothroyd, J. C. 1993; 262 (5135): 911-914

  Abstract

  A system for stable transformation of Toxoplasma gondii tachyzoites was developed that exploited the susceptibility of Toxoplasma to chloramphenicol. Introduction of the chloramphenicol acetyltransferase (CAT) gene fused to Toxoplasma flanking sequences followed by chloramphenicol selection resulted in parasites stably expressing CAT. A construct incorporating the tandemly repeated gene, B1, targeted efficiently to its homologous chromosomal locus. Knockout of the single-copy gene, ROP1, was also successful. Stable transformation should permit the identification and analysis of Toxoplasma genes important in the interaction of this opportunistic parasite with its host.

  View details for Web of Science ID A1993MF43800040

  View details for PubMedID 8235614

• DEVELOPMENT OF GENETIC SYSTEMS FOR TOXOPLASMA-GONDII PARASITOLOGY TODAY Sibley, L. D., Pfefferkorn, E. R., Boothroyd, J. C. 1993; 9 (10): 392-395

  Abstract

  The protozoan parasite Toxoplasma gondii has recently emerged as an important opportunistic pathogen in humans. Toxoplasma also shares a number of biological features with Plasmodium and Eimeria, which are important pathogens of humans and animals. Because o f the ease o f experimental use, David Sibley, Elmer Pfefferkom and John Boothroyd have undertaken the development of genetics in Toxoplasma as a model intracellular parasite. Toxoplasma is presently the only parasitic protozoan where both classical and molecular genetics are feasible. The recent advances in this system are highlighted here, along with potential applications of genetics for understanding intracellular parasitism.

  View details for Web of Science ID A1993LX69300016

  View details for PubMedID 15463677

• EXPRESSION OF TOXOPLASMA-GONDII P-30 AS FUSIONS WITH GLUTATHIONE-S-TRANSFERASE IN ANIMAL-CELLS BY SINDBIS RECOMBINANT VIRUS MOLECULAR AND BIOCHEMICAL PARASITOLOGY Xiong, C., Grieve, R. B., Kim, K., Boothroyd, J. C. 1993; 61 (1): 143-148

  View details for Web of Science ID A1993LZ79600015

  View details for PubMedID 8259126

• MOLECULAR-CLONING AND CELLULAR-LOCALIZATION OF A BIP HOMOLOG IN TRYPANOSOMA-BRUCEI - DIVERGENT ER RETENTION SIGNALS IN A LOWER EUKARYOTE JOURNAL OF CELL SCIENCE Bangs, J. D., Uyetake, L., BRICKMAN, M. J., Balber, A. E., Boothroyd, J. C. 1993; 105: 1101-1113

  Abstract

  Using the polymerase chain reaction with degenerate primers, three new members of the hsp70 gene family of Trypanosoma brucei have been identified. A genomic clone of one of these, gA, has been fully sequenced and the corresponding gene product has been characterized using antibody to recombinant gA fusion protein. gA is the trypanosomal homologue of BiP, an endoplasmic reticulum resident hsp70 gene family member, based on four lines of evidence: (1) gA protein has 64% deduced amino acid identity with rat BiP; (2) the deduced amino acid sequence has a putative secretory signal peptide; (3) the gA gene product is a soluble luminal resident of a trypanosomal microsome fraction; (4) the gA polypeptide does not cofractionate with mitochondrial markers. Trypanosomes are the most primitive eukaryote yet in which BiP has been identified. The gA polypeptide has been used as a specific marker for the direct visualization of endoplasmic reticulum in trypanosomes by both indirect immunofluorescence and cryoimmuno electron microscopy. The endoplasmic reticulum is seen as a tubular network that extends throughout the cell excluding the flagellum. The C-terminal tetrapeptide of gA is MDDL, which, together with the C-terminal tetrapeptide (KQDL) of a trypanosome protein disulfide isomerase homologue (Hsu et al. (1989) Biochemistry 28, 6440-6446), indicates that endoplasmic reticulum retrieval signals in trypanosomes may be as divergent and heterogeneous as any seen in the other eukaryotes yet studied.

  View details for Web of Science ID A1993LV68700024

  View details for PubMedID 8227199

• TRANSIENT TRANSFECTION AND EXPRESSION IN THE OBLIGATE INTRACELLULAR PARASITE TOXOPLASMA-GONDII SCIENCE Soldati, D., Boothroyd, J. C. 1993; 260 (5106): 349-352

  Abstract

  Toxoplasma gondii is a protozoan pathogen that produces severe disease in humans and animals. This obligate intracellular parasite provides an excellent model for the study of how such pathogens are able to invade, survive, and replicate intracellularly. DNA encoding chloramphenicol acetyltransferase was introduced into T. gondii and transiently expressed with the use of three vectors based on different Toxoplasma genes. The ability to introduce genes and have them efficiently and faithfully expressed is an essential tool for understanding the structure-function relation of genes and their products.

  View details for Web of Science ID A1993KX80000037

  View details for PubMedID 8469986

• POPULATION BIOLOGY OF TOXOPLASMA - CLONALITY, VIRULENCE, AND SPECIATION (OR NOT) INFECTIOUS AGENTS AND DISEASE-REVIEWS ISSUES AND COMMENTARY Boothroyd, J. C. 1993; 2 (2): 100-102

  Abstract

  The population biology of Toxoplasma gondii is reviewed in the context of how its complex life cycle might be responsible for the current picture and how a knowledge of the population structure may have an impact on the thinking of both clinician and researcher. Published results already clearly indicate that the population can be divided into two distinct groups that are genetically and biologically distinct. The more virulent group, as defined by their LD50 in mice, is apparently a single, clonal line. The biological and clinical implications of the existence of such a group are discussed.

  View details for Web of Science ID A1993LH70400004

  View details for PubMedID 8162354

• STABLE TRANSFORMATION OF THE OPPORTUNISTIC PATHOGEN TOXOPLASMA USING CHLORAMPHENICOL SELECTION Kim, K., Boothroyd, J. C. SLACK INC. 1993: A209–A209

  View details for Web of Science ID A1993KW76100527

• POPULATION BIOLOGY OF TOXOPLASMA-GONDII RESEARCH IN IMMUNOLOGY Boothroyd, J. C., Sibley, L. D. 1993; 144 (1): 14-16

  View details for Web of Science ID A1993KK61300003

  View details for PubMedID 8451513

• GENERATION OF A RESTRICTION-FRAGMENT-LENGTH-POLYMORPHISM LINKAGE MAP FOR TOXOPLASMA-GONDII GENETICS Sibley, L. D., LeBlanc, A. J., Pfefferkorn, E. R., Boothroyd, J. C. 1992; 132 (4): 1003-1015

  Abstract

  We have constructed a genetic linkage map for the parasitic protozoan, Toxoplasma gondii, using randomly selected low copy number DNA markers that define restriction fragment length polymorphisms (RFLPs). The inheritance patterns of 64 RFLP markers and two phenotypic markers were analyzed among 19 recombinant haploid progeny selected from two parallel genetic crosses between PLK and CEP strains. In these first successful interstrain crosses, these RFLP markers segregated into 11 distinct genetic linkage groups that showed close correlation with physical linkage groups previously defined by molecular karyotype. Separate linkage maps, constructed for each of the 11 chromosomes, indicated recombination frequencies range from approximately 100 to 300 kb per centimorgan. Preliminary linkage assignments were made for the loci regulating sinefungin resistance (snf-1) on chromosome IX and adenine arabinoside (ara-1) on chromosome V by linkage to RFLP markers. Despite random segregation of separate chromosomes, the majority of chromosomes failed to demonstrate internal recombination events and in 3/19 recombinant progeny no intramolecular recombination events were detected. The relatively low rate of intrachromosomal recombination predicts that tight linkage for unknown genes can be established with a relatively small set of markers. This genetic linkage map should prove useful in mapping genes that regulate drug resistance and other biological phenotypes in this important opportunistic pathogen.

  View details for Web of Science ID A1992JZ83200013

  View details for PubMedID 1360931

• GENES THAT ALLOW YEAST-CELLS TO GROW IN THE ABSENCE OF THE HDEL RECEPTOR EMBO JOURNAL Hardwick, K. G., Boothroyd, J. C., Rudner, A. D., Pelham, H. R. 1992; 11 (11): 4187-4195

  Abstract

  The ERD2 gene of Saccharomyces cerevisiae encodes the HDEL receptor that sorts ER proteins; it is essential for growth. In the absence of Erd2p the Golgi apparatus is both functionally and morphologically perturbed. Here we describe the isolation of four SED genes (suppressors of the erd2-deletion) which, when present in multiple copies, allow cells to grow in the absence of ERD2. The suppressed strains secrete the ER protein BiP and their internal membranes show a variety of morphological abnormalities. Sequence analysis indicates that all these SED genes encode membrane proteins: SED1 encodes a probable cell surface glycoprotein; SED2 is identical to SEC12, a gene required for the formation of ER-derived transport vesicles; SED4 encodes a protein whose cytoplasmic domain is 45% identical to that of Sec12p; SED3 is DPM1, the structural gene for dolichol-P-mannose synthase. We suggest that the absence of ERD2 causes an imbalance between membrane flow into and out of the Golgi apparatus, and that the SED gene products can compensate for this either by slowing transport from the ER or by stimulating vesicle budding from Golgi membranes.

  View details for Web of Science ID A1992JT32800041

  View details for PubMedID 1327759

• VIRULENT-STRAINS OF TOXOPLASMA-GONDII COMPRISE A SINGLE CLONAL LINEAGE NATURE Sibley, L. D., Boothroyd, J. C. 1992; 359 (6390): 82-85

  Abstract

  The protozoan Toxoplasma gondii is a prevalent parasite in wild and domestic animals worldwide, being transmitted through the food chain by carnivorous feeding and scavenging. Toxoplasma normally divides asexually to yield a haploid form that can infect virtually any vertebrate but it also has a well defined sexual cycle that occurs exclusively in cats. Toxoplasma has become important as an often fatal opportunistic pathogen in patients with AIDS, although the 15-85% of adult human populations that are chronically infected with T. gondii are typically asymptomatic. Infections in immunocompromised hosts have variable outcomes. For example, only 30 to 50% of AIDS patients that are chronically infected with the parasite develop toxoplasmic encephalitis and only about half of acute maternal infections result in congenital disease of the newborn. T. gondii strains differ in their virulence in animals, but the extent to which different strains are related has not been determined. Here we analyse 28 strains from a variety of hosts on five continents and find that the ten virulent strains have an essentially identical genotype, whereas the nonvirulent strains are moderately polymorphic. These data strongly suggest that virulent strains of T. gondii originated from a single lineage which has remained genetically homogeneous despite being globally widespread, and despite the ability of this organism to reproduce sexually.

  View details for Web of Science ID A1992JL66200060

  View details for PubMedID 1355855

• GENOMIC ORGANIZATION AND CONTEXT OF A TRYPANOSOME VARIANT SURFACE GLYCOPROTEIN GENE FAMILY JOURNAL OF MOLECULAR BIOLOGY Beals, T. P., Boothroyd, J. C. 1992; 225 (4): 961-971

  Abstract

  We have defined the genomic organization and genomic context of a Trypanosoma brucei brucei gene family encoding variant surface glycoproteins (VSGs). This gene family is neither tandemly repeated nor closely linked in the genome, and is not located on small or intermediate size chromosomes. Two dispersed repeated sequence elements, RIME-ingi and the upstream repeat sequence, are linked to members of this gene family; however, the upstream repeat sequences are closely linked only to the basic copy. In other isolates of T.b. brucei this gene family appears conserved with some variation; a restriction fragment length polymorphism found among these isolates suggests the hypothesis that VSG genes may occasionally be diploid. A model accounting for both the generation of dispersed families of VSG genes, and for the interstrain variability of VSG genes, is proposed.

  View details for Web of Science ID A1992JA43700006

  View details for PubMedID 1613802

• SEQUENCE DIVERGENCE AMONG MEMBERS OF A TRYPANOSOME VARIANT SURFACE GLYCOPROTEIN GENE FAMILY JOURNAL OF MOLECULAR BIOLOGY Beals, T. P., Boothroyd, J. C. 1992; 225 (4): 973-983

  Abstract

  We have used analysis of DNA sequence data from four members of a Trypanosoma brucei variant surface glycoprotein gene family to investigate the molecular basis of the generation of antigenic diversity in African trypanosomes. Among these four sequences we find the greatest similarity in the untranslated sequences immediately upstream from the coding region. A complex pattern of nucleic acid and predicted amino acid sequence divergence appears starting at the coding sequence. Two related but highly divergent hydrophobic leaders are associated with different members of this gene family; both forms of these hydrophobic leaders appear to exist in other isolates of T. b. brucei. We find conservative replacements in the first 120 predicted amino acid residues of the mature protein; the following 80 predicted residues show less conservative replacements, and we suggest that this region may be hypervariable and exposed to the aqueous environment.

  View details for Web of Science ID A1992JA43700007

  View details for PubMedID 1613803

• MASS-SPECTROMETRY OF MESSENGER-RNA CAP-4 FROM TRYPANOSOMATIDS REVEALS 2 NOVEL NUCLEOSIDES JOURNAL OF BIOLOGICAL CHEMISTRY Bangs, J. D., Crain, P. F., Hashizume, T., MCCLOSKEY, J. A., Boothroyd, J. C. 1992; 267 (14): 9805-9815

  Abstract

  Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.

  View details for Web of Science ID A1992HT96500058

  View details for PubMedID 1349605

• CONSTRUCTION OF A MOLECULAR KARYOTYPE FOR TOXOPLASMA-GONDII MOLECULAR AND BIOCHEMICAL PARASITOLOGY Sibley, L. D., Boothroyd, J. C. 1992; 51 (2): 291-300

  Abstract

  As an important step in developing genetic systems for the protozoan parasite Toxoplasma gondii, we have constructed a molecular karyotype based on separation of chromosomes by pulsed field gel electrophoresis and assignment of linkage groups by hybridization. Toxoplasma chromosomes were separated using transverse alternating field electrophoresis (TAFE) gels into 9 distinct bands that defined a minimum of 10 physical linkage groups with apparent sizes that range from approximately 2 Mb to more than 6 Mb. Individual chromosome sizes were stable with prolonged mitotic passage of a single strain but varied by approximately 15% for chromosomes III and V between three different strains of Toxoplasma. Preliminary physical linkage groups were defined by mapping 57 single or low copy number probes to specific chromosomes by hybridization. The majority of these probes consist of random DNA segments; however, a number of cDNAs encoding important structural and antigenic components were also mapped to specific linkage groups. Assuming random distribution, this set of probes should provide approximately 1 marker every 1.0-1.5 Mb over the 80 Mb haploid genome and should greatly aid in using genetics to study the biology, drug resistance, and virulence of this important opportunistic pathogen.

  View details for Web of Science ID A1992HK16900013

  View details for PubMedID 1574087

• ANTIGEN-SPECIFIC (P-30) MOUSE CD8+ T-CELLS ARE CYTOTOXIC AGAINST TOXOPLASMA-GONDII-INFECTED PERITONEAL-MACROPHAGES JOURNAL OF IMMUNOLOGY Kasper, L. H., Khan, I. A., Ely, K. H., Buelow, R., Boothroyd, J. C. 1992; 148 (5): 1493-1498

  Abstract

  The importance of CD8+ T cells in immunity against Toxoplasma gondii is now well recognized. The mechanism by which these CD8+ T cells are able to confer this immunity is not yet understood. To examine the Ag specificity of this response, immune splenocytes from mice immunized with p30, a major surface parasite Ag, were evaluated for their ability to lyse peritoneal macrophages infected with three different strains of T. gondii. Macrophages infected with either the RH or P wild-type strain tachyzoites were lysed at varying E:T ratios by nylon wool nonadherent immune splenocytes whereas macrophages infected with a p30-deficient mutant (B mutant) of the P strain were not. The gene encoding p30 for the wild type and B mutant were amplified by the polymerase chain reaction. This revealed a nonsense mutation in the B mutant such that its primary translation product is predicted to be about two-thirds the size of the wild-type p30 molecule. mAb depletion studies indicate that the cytotoxic effect of the immune splenocytes is mediated by the CD8+ T cell population. Peritoneal macrophages infected with the three different strains (RH, P wild type, B mutant) from mice genetically restricted were not lysed by the immune CD8+ effector cell population. A cloned line (C3) of p30 Ag-specific CD8+ T cells exhibited significant cytotoxicity against syngeneic peritoneal macrophages infected with either the RH or P strain tachyzoites. There was no macrophage lysis observed by these CD8+ effector cells of either syngeneic macrophages infected with the B mutant or nonsyngeneic macrophages infected with the three different tachyzoite strains.

  View details for Web of Science ID A1992HF26600030

  View details for PubMedID 1538132

• A TOXOPLASMA-GONDII RHOPTRY PROTEIN ASSOCIATED WITH HOST-CELL PENETRATION HAS UNUSUAL CHARGE ASYMMETRY MOLECULAR AND BIOCHEMICAL PARASITOLOGY Ossorio, P. N., Schwartzman, J. D., Boothroyd, J. C. 1992; 50 (1): 1-16

  Abstract

  The monoclonal antibody Tg49 both recognizes a Toxoplasma gondii rhoptry protein (ROP1) and inhibits penetration enhancing factor. The latter is a proteinaceous factor found in Toxoplasma lysates or conditioned media that increases the efficiency with which parasites invade host cells. Tg49 was used to screen a lambda gt11 cDNA library and the clone obtained was identified as the cognate gene for ROP1 by several criteria: (1) recombinant protein reacted with the monoclonal; (2) antiserum against the recombinant reacted with the same bands on Western blots as did Tg49; and (3) antiserum against the recombinant recognized a protein in the rhoptries. The ROP1 gene is a single copy gene with a message of approximately 2.1 kb. The predicted polypeptide sequence of ROP1 shows an unusual charge and amino acid asymmetry. There is a highly acidic, proline-rich domain in the amino-terminal portion of the predicted protein, followed by a strongly basic carboxy-terminal domain. An octapeptide repeat is found almost midway through the peptide sequence toward the end of the acidic domain. The ROP1 gene was expressed in a bacterial system, and the resulting polypeptide exhibited anomalous migration on polyacrylamide gel electrophoresis. Given that Tg49 inhibits penetration enhancing factor, it seems likely that the ROP1 protein is a component of that factor, and that the unusual sequence of this protein plays some role in host cell penetration by T. gondii.

  View details for Web of Science ID A1992GW36200001

  View details for PubMedID 1542304

• MITOCHONDRIAL-LIKE DNA-SEQUENCES FLANKED BY DIRECT AND INVERTED REPEATS IN THE NUCLEAR GENOME OF TOXOPLASMA-GONDII JOURNAL OF MOLECULAR BIOLOGY Ossorio, P. N., Sibley, L. D., Boothroyd, J. C. 1991; 222 (3): 525-536

  Abstract

  In the course of our genetic studies on Toxoplasma gondii, it was discovered that one cosmid hybridized to a repetitive element. The hybridization pattern observed for the enzyme BglII indicated that this cosmid hybridized to a large number of discrete, but related elements. Four BglII fragments were subcloned from the cosmid, and each was shown to hybridize with all the others, as well as to numerous dispersed sequences in genomic DNA. Three subclones were sequenced in their entirety, and shown to contain fragments of the genes for cytochrome oxidase subunit I and apocytochrome b, complete and functional copies of which have been found in only mitochondrial genomes. All the subcloned fragments were bounded at both ends by a 91 base-pair sequence, which contains a site for BglII. This 91 base-pair sequence could be found as either a direct or inverted repeat. It was determined that the BglII elements are arrayed downstream from a single copy nuclear gene. Comparison of genomic and cosmid DNAs confirmed that the cosmid faithfully reflects the nuclear genome. Although the mitochondrial genome of Toxoplasma has not been characterized, these nuclear mitochondrial-like sequences appear to be internally rearranged with respect to known, functional mitochondrial genomes, and with respect to each other. The finding of short repeated sequences flanking these elements may be a clue to the mechanism of their dissemination.

  View details for Web of Science ID A1991GV58300012

  View details for PubMedID 1660924

• PROPOSAL FOR A UNIFORM GENETIC NOMENCLATURE IN TOXOPLASMA-GONDII PARASITOLOGY TODAY Sibley, L. D., Pfefferkorn, E. R., Boothroyd, J. C. 1991; 7 (12): 327-328

  View details for Web of Science ID A1991GW10900003

  View details for PubMedID 15463406

• PROTECTION OF MICE FROM FATAL TOXOPLASMA-GONDII INFECTION BY IMMUNIZATION WITH P-30 ANTIGEN IN LIPOSOMES JOURNAL OF IMMUNOLOGY Bulow, R., Boothroyd, J. C. 1991; 147 (10): 3496-3500

  Abstract

  Using liposomes as adjuvant, a purified membrane Ag from Toxoplasma gondii (p30) has been tested for its protective effect in mice. Immunization with p30 in liposomes resulted in only one in 15 mice dying from a challenge that killed 11 of 15 control mice (receiving only buffer or liposomes without p30). The p30 Ag alone gave intermediate levels of protection, with 5 of 15 animals dying. The source of the p30 Ag was the rapidly growing, laboratory-adapted strain of T. gondii, RH; challenge was with the recently isolated C strain which still has all the properties of a wild-type strain. To assess the validity of this combination, the amino acid sequence of p30 from these two strains (as predicted from the corresponding gene sequence) was compared and found to differ in only eight residues. This minimal variation argued that RH was a valid source of material for a subunit vaccine, as subsequently confirmed by the protection studies. These results indicate that p30 is an appropriate Ag for development into a subunit vaccine for immunization of humans and/or domestic livestock, which are a major source of human infection.

  View details for Web of Science ID A1991GQ17100036

  View details for PubMedID 1940349

• INHIBITION OF PROTEIN-SYNTHESIS RESULTS IN SUPER-INDUCTION OF PROCYCLIN (PARP) RNA LEVELS MOLECULAR AND BIOCHEMICAL PARASITOLOGY Dorn, P. L., Aman, R. A., Boothroyd, J. C. 1991; 44 (1): 133-139

  Abstract

  Procyclin is an abundant surface antigen found exclusively on the procyclic forms of African trypanosomes. We are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. We find that increased levels of procyclin RNA are evident as early as 15 min after triggering differentiation. The increase in procyclin RNA levels requires the temperature shift from 37 degrees C to 27 degrees C and is aided by addition of the tricarboxylic acid cycle intermediate cis-aconitate. Maximal induction is observed with a combination of three triggers of differentiation: citrate, cis-aconitate and the temperature shift. Protein synthesis does not appear to be required for induction of procyclin RNA during differentiation. In fact, addition of protein synthesis inhibitors results in super-induction of procyclin RNA levels, even under conditions where no induction is normally observed (i.e., at 37 degrees C in the absence of citrate and cis-aconitate). This super-induction was observed with four different protein synthesis inhibitors that affect different stages of translation. Thus, the accumulation of procyclin transcripts may be under the control of a negative regulator whose effective levels are reduced during differentiation from bloodstream to procyclic forms.

  View details for Web of Science ID A1991EM82100015

  View details for PubMedID 2011148

• AN EXPRESSION-SITE-ASSOCIATED GENE FAMILY OF TRYPANOSOMES IS EXPRESSED INVIVO AND SHOWS HOMOLOGY TO A VARIANT SURFACE GLYCOPROTEIN GENE MOLECULAR AND BIOCHEMICAL PARASITOLOGY Hobbs, M. R., Boothroyd, J. C. 1990; 43 (1): 1-16

  Abstract

  Utilizing first-strand cDNA from different stages, a gene family was identified that is expressed in bloodstream form trypanosomes but not in cultured procyclic forms. This family of 50-100 genes, termed bloodstream-specific 1 (BS1), shares a chromosomal distribution pattern similar to the variant surface glycoprotein (VSG) genes and the expression-site-associated genes (ESAGs). The BS1 genes are expressed in several variants of Trypanosoma brucei brucei and in Trypanosoma brucei gambiense. Sequence analysis of five members of this gene family reveals the recently described ESAG 6 and ESAG 7 genes as well as the ESAG X gene to be members of this family. We have been unable to localize the BS1 gene product in the cell but show that chronically infected rabbit serum recognizes recombinant BS1 protein arguing for expression in vivo. Finally we note that the derived protein sequence for the BS1 genes suggests an evolutionary relationship with at least one variant surface glycoprotein gene, and hence these studies may provide clues to understanding the molecular origins of antigenic variation in trypanosomes.

  View details for Web of Science ID A1990ED88100001

  View details for PubMedID 2290439

• RAPID PRENATAL-DIAGNOSIS OF CONGENITAL TOXOPLASMA INFECTION BY USING POLYMERASE CHAIN-REACTION AND AMNIOTIC-FLUID JOURNAL OF CLINICAL MICROBIOLOGY GROVER, C. M., Thulliez, P., Remington, J. S., Boothroyd, J. C. 1990; 28 (10): 2297-2301

  Abstract

  Infection of pregnant women with Toxoplasma gondii places the developing fetus at risk for congenital infection. We report a prospective study of 43 documented cases of acute maternal Toxoplasma infections acquired during gestation in which the polymerase chain reaction (PCR) was evaluated for diagnosis of fetal infection and compared with the current standard methods. On the basis of direct lysis of pelleted amniotic fluid cells followed by amplification of a gene sequence specific for T. gondii, PCR correctly identified the presence of T. gondii in five of five samples of amniotic fluid from four proven cases of congenital infection. PCR also detected three of five positive cases from a nonprospective group. The two diagnostic methods of comparable speed, detection of specific immunoglobulin M from fetal blood and and inoculation of amniotic fluid into tissue culture, correctly identified only 3 and 4 of the 10 positive samples, respectively. The considerably more time-consuming methods of mouse inoculation of amniotic fluid and fetal blood both detected 7 of 10 positive samples. There were no false-positive diagnoses by any of the methods. Therefore, detection of T. gondii by PCR appears to be the most promising method for prenatal diagnosis of congenital Toxoplasma infection, since it is both extremely rapid and highly sensitive.

  View details for Web of Science ID A1990DZ42500028

  View details for PubMedID 2229355

• LACK OF INTRONS IN THE RIBOSOMAL-PROTEIN GENE S14 OF TRYPANOSOMES MOLECULAR AND CELLULAR BIOLOGY Perelman, D., Boothroyd, J. C. 1990; 10 (6): 3284-3288

  Abstract

  Introns are almost always present in ribosomal protein genes, even in organisms in which introns are rare. Although trans spliced, the trypanosome ribosomal protein gene S14 apparently does not have cis introns, which supports the notion that such introns are absent in this organism.

  View details for Web of Science ID A1990DF42500101

  View details for PubMedID 2188114

• HEAT-SHOCK DISRUPTION OF TRANS-SPLICING IN TRYPANOSOMES - EFFECT ON HSP70, HSP85 AND TUBULIN MESSENGER-RNA SYNTHESIS GENE MUHICH, M. L., Hsu, M. P., Boothroyd, J. C. 1989; 82 (1): 169-175

  Abstract

  The synthesis of most, if not all, mRNA in trypanosomes involves the splicing in trans of two RNA molecules. One of these includes the common 5' mini-exon or spliced-leader sequence, while the other contains the complete protein-coding exon sequence for a given gene. We have recently shown that trans-splicing of tubulin transcripts is disrupted by heat shock, whereas trans-splicing of Hsp70 mRNA continues unabated, despite the increased expression of this latter gene [Muhich and Boothroyd, Mol. Cell Biol. 8 (1988) 3837-3846]. In this report we show that, in addition to being polyadenylated, the dimeric beta-alpha tubulin transcripts which accumulate as a result of heat shock have a correctly processed 5'-end (through mini-exon addition), and that the extent of disruption is proportional to the degree of heat shock. We further show that mRNA levels for two evolutionarily-conserved heat-shock proteins, Hsp70 and Hsp85, are increased under the splicing-disruptive conditions of heat shock.

  View details for Web of Science ID A1989AU55800018

  View details for PubMedID 2684772

• DIRECT AND SENSITIVE DETECTION OF A PATHOGENIC PROTOZOAN, TOXOPLASMA-GONDII, BY POLYMERASE CHAIN-REACTION JOURNAL OF CLINICAL MICROBIOLOGY Burg, J. L., GROVER, C. M., Pouletty, P., Boothroyd, J. C. 1989; 27 (8): 1787-1792

  Abstract

  We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (10(5)/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.

  View details for Web of Science ID A1989AG26500016

  View details for PubMedID 2768467

• A DEVELOPMENTALLY REGULATED GENE OF TRYPANOSOMES ENCODES A HOMOLOG OF RAT PROTEIN-DISULFIDE ISOMERASE AND PHOSPHOINOSITOL PHOSPHOLIPASE-C BIOCHEMISTRY Hsu, M. P., MUHICH, M. L., Boothroyd, J. C. 1989; 28 (15): 6440-6446

  Abstract

  We have isolated and characterized a developmentally regulated gene in Trypanosoma brucei, arbitrarily termed BS2. BS2 mRNA is substantially more abundant in bloodstream-form trypanosomes than in procyclic culture forms. Its nucleotide sequence reveals a single contiguous open-reading frame of 497 codons and is predicted to encode a protein of approximately 55.5 kilodaltons. A search of the NBRF protein data base revealed that within the predicted amino acid sequence are two of the evolutionarily conserved redox sites typified by thioredoxin of bacteria. Of this family of proteins, the recently sequenced rat genes encoding protein-disulfide isomerase (PDI) and form I phosphoinositide-specific phospholipase C (PIPLC) showed homology extending over the length of all three proteins (i.e., between BS2, PDI, and PIPLC). Although this homology includes the acidic C-terminus characteristic of proteins localized to the lumen of the endoplasmic reticulum, the BS2 product is predicted to possess multiple sites for N-linked glycosylation while PDI and PIPLC have none. Possible roles of the BS2 gene product in trypanosome physiology are discussed.

  View details for Web of Science ID A1989AH82900041

  View details for PubMedID 2551375

• SYNTHESIS OF TRYPANOSOME HSP70 MESSENGER-RNA IS RESISTANT TO DISRUPTION OF TRANS-SPLICING BY HEAT-SHOCK JOURNAL OF BIOLOGICAL CHEMISTRY MUHICH, M. L., Boothroyd, J. C. 1989; 264 (13): 7107-7110

  Abstract

  Synthesis of mRNA in trypanosomes involves an apparent trans-splicing reaction whereby a common 39-nucleotide mini-exon sequence is joined to the protein-coding exon of a mRNA precursor. We have previously shown (Muhich, M. L., and Boothroyd, J. C. (1988) Mol. Cell. Biol. 8, 3837-3846) that the trans-splicing pathway of Trypanosoma brucei is sensitive to disruption by severe heat shock. Here we demonstrate that the synthesis of heat shock protein 70 (hsp 70) mRNA in T. brucei is apparently resistant to the heat-induced disruption of splicing. The 5'-ends of hsp 70 mRNAs are shown to be identical for molecules synthesized at either normal or heat shock temperatures, and in both cases, the 5'-terminal mini-exon sequence is present. These results suggest that T. brucei has evolved a mechanism which directly compensates for the deleterious effects of heat shock on trans-splicing, one which allows for the continued mini-exon-dependent trans-splicing of selected pre-mRNAs.

  View details for Web of Science ID A1989U326600007

  View details for PubMedID 2708359

• THE MAJOR SURFACE-ANTIGEN, P-30, OF TOXOPLASMA-GONDII IS ANCHORED BY A GLYCOLIPID JOURNAL OF BIOLOGICAL CHEMISTRY NAGEL, S. D., Boothroyd, J. C. 1989; 264 (10): 5569-5574

  Abstract

  P30, the major surface antigen of the parasitic protozoan Toxoplasma gondii, can be specifically labeled with [3H]palmitic acid and with myo-[2-3H]inositol. The fatty acid label can be released by treatment of P30 with phosphatidylinositol-specific phospholipase C (PI-PLC). Such treatment exposes an immunological "cross-reacting determinant" first described on Trypanosoma brucei variant surface glycoprotein. PI-PLC cleavage of intact parasites metabolically labeled with [35S]methionine results in the release of intact P30 polypeptide in a form which migrates faster in polyacrylamide gel electrophoresis. These results argue that P30 is anchored by a glycolipid. Results from thin layer chromatography analysis of purified [3H] palmitate-labeled P30 treated with PI-PLC, together with susceptibility to mild alkali hydrolysis and to cleavage with phospholipase A2, suggest that the glycolipid anchor of T. gondii P30 includes a 1,2-diacylglycerol moiety.

  View details for Web of Science ID A1989T999200041

  View details for PubMedID 2925621

• CLONING OF CDNAS ENCODING A 28 KILODALTON ANTIGEN OF TOXOPLASMA-GONDII MOLECULAR AND BIOCHEMICAL PARASITOLOGY Prince, J. B., Araujo, F. G., Remington, J. S., Burg, J. L., Boothroyd, J. C., Sharma, S. D. 1989; 34 (1): 3-13

  Abstract

  By screening cDNA libraries in lambda gt11 with antibodies raised against the previously described protective F3G3 antigen of Toxoplasma gondii, and subsequently screening with nucleic acid probes, we have isolated cDNA clones that encode a 28 kDa antigen of T. gondii that is likely one of the two antigenic components of the F3G3 antigen. The gene apparently exists as a single copy in the tachyzoite haploid genome of the three strains of T. gondii examined. Northern blot analyses revealed that the cDNAs hybridize with a major T. gondii RNA species of 1.1 kb. Together the cDNAs encompass 1051 bp of cDNA sequence containing an open reading frame with the capacity to encode a 28 kDa protein. Antibodies that were affinity purified using recombinant fusion proteins produced by two of the clones reacted on protein blots of whole T. gondii lysate with a single antigen having an apparent molecular mass of 28 kDa. Both recombinant fusion proteins reacted with IgG antibodies in sera of mice and humans infected with T. gondii and therefore might be useful for the development of diagnostic assays for T. gondii infection.

  View details for Web of Science ID A1989T815000001

  View details for PubMedID 2710166

• MOLECULAR ANALYSIS OF THE GENE ENCODING THE MAJOR SURFACE-ANTIGEN OF TOXOPLASMA-GONDII JOURNAL OF IMMUNOLOGY Burg, J. L., Perelman, D., Kasper, L. H., Ware, P. L., Boothroyd, J. C. 1988; 141 (10): 3584-3591

  Abstract

  The complete sequence of P30, the major surface Ag of the protozoan parasite, Toxoplasma gondii, has been deduced through the cloning and analysis of its gene. Using polyclonal serum specific for P30, we have isolated a P30 cDNA clone from a lambda gt11 cDNA expression library derived from tachyzoites of T. gondii (RH strain). This clone produces a beta-galactosidase fusion protein which reacts with several anti-P30 mAb. In addition, polyclonal anti-serum raised to the fusion protein reacts with purified P30 protein and exclusively with P30 in a whole cell lysate of T. gondii. This cDNA clone was used to isolate near full-length cDNA molecules and a cosmid clone containing the P30 gene. Sequence analysis of the cDNA reveals a single open reading frame with coding capacity for 34.7 kDa of primary translation product (consistent with the apparent Mr of P30 on SDS-acrylamide gels) including a presumptive hydrophobic signal sequence. The P30 primary translation product also has a carboxy-terminal hydrophobic tail which is predictive of a posttranslational cleavage and modification with a glycolipid anchor. We have identified the apparent 5' and 3' ends of the P30 mRNA transcript which is extremely abundant, 1500 nucleotides in length, and polyadenylated. The P30 gene is single copy and contains no introns.

  View details for Web of Science ID A1988Q824700051

  View details for PubMedID 3183382

• POLYCISTRONIC TRANSCRIPTS IN TRYPANOSOMES AND THEIR ACCUMULATION DURING HEAT-SHOCK - EVIDENCE FOR A PRECURSOR ROLE IN MESSENGER-RNA SYNTHESIS MOLECULAR AND CELLULAR BIOLOGY MUHICH, M. L., Boothroyd, J. C. 1988; 8 (9): 3837-3846

  Abstract

  Maturation of mRNA precursors in trypanosomes involves an apparent trans splicing event in which a 39-nucleotide miniexon sequence, common to all trypanosome mRNAs, is joined to the 5' end of a protein-coding exon. We demonstrate that the processing machinery responsible for the maturation of tubulin mRNA precursors in Trypanosoma brucei can be disrupted by heat shock. This results in an accumulation of polycistronic RNA species and a decrease in the abundance of branched splicing intermediates. At normal temperatures, tubulin polycistronic transcripts were also detected and were shown in pulse-chase experiments to be abundantly synthesized and very rapidly turned over. These results, combined with results of the heat shock experiments, suggest that these polycistronic transcripts are the precursors of the (monocistronic) tubulin mRNAs of trypanosomes.

  View details for Web of Science ID A1988P854100031

  View details for PubMedID 3221866

• THE ALPHA-TUBULINS AND BETA-TUBULINS OF TOXOPLASMA-GONDII ARE ENCODED BY SINGLE COPY GENES CONTAINING MULTIPLE INTRONS MOLECULAR AND BIOCHEMICAL PARASITOLOGY NAGEL, S. D., Boothroyd, J. C. 1988; 29 (2-3): 261-273

  Abstract

  We have studied the fundamentals of gene expression in the protozoan parasite Toxoplasma gondii by analyzing, in detail, the genes encoding alpha- and beta-tubulin. Southern analysis and quantitation studies reveal that, unlike in other organisms studied thus far, both these genes are present as single copies in the haploid Toxoplasma genome. Sequencing of these genes indicates that they both contain multiple introns with conserved 5' and 3' splice site signals. We have found that HeLa cell nuclear extracts are able to accurately splice a Toxoplasma pre-mRNA construct. We have mapped, for the alpha-tubulin gene, the exact site of transcription initiation and the approximate site of poly A addition by primer extension and RNase protection assays. Trans-splicing, as demonstrated in the Kinetoplastida, is not involved in the formation of the mature alpha-tubulin transcript in T. gondii.

  View details for Web of Science ID A1988N920500017

  View details for PubMedID 3412377

• TRYPANOSOME TRANS-SPLICING UTILIZES 2'-5' BRANCHES AND A CORRESPONDING DEBRANCHING ACTIVITY EMBO JOURNAL Sutton, R. E., Boothroyd, J. C. 1988; 7 (5): 1431-1437

  Abstract

  The 5' ends of trypanosome mRNAs consist of an identical sequence of 39 nucleotides which is derived from a discrete transcript of approximately 140 nucleotides (medRNA). It has been proposed that generation of chimeric mRNAs in trypanosomes occurs by the process of trans-splicing involving medRNA and an acceptor RNA. Part of the basis for this suggestion comes from the ability of HeLa cell extracts (known to contain debranching activity) to catalyze the release of the intron portion of medRNA (minRNA) implying a Y-branched intermediate in the splicing process. Here we provide direct chemical analysis that miniRNA is attached to higher mol. wt RNA molecules by a 2'-5' phosphodiester bond (i.e. as a branched structure). We also demonstrate that trypanosomes have substantial amounts of debranching activity which is similar in nature to that of HeLa cells. These results provide further evidence for trans-splicing in trypanosomes and highlights its similarity to cis-splicing in other eukaryotes.

  View details for Web of Science ID A1988N282500024

  View details for PubMedID 3409870

• THE CAP OF BOTH MINIEXON-DERIVED RNA AND MESSENGER-RNA OF TRYPANOSOMES IS 7-METHYLGUANOSINE MOLECULAR AND CELLULAR BIOLOGY Sutton, R. E., Boothroyd, J. C. 1988; 8 (1): 494-496

  Abstract

  Most, if not all, trypanosome mRNAs have the same 35-base sequence at their 5' terminus which is derived from a short RNA (medRNA) probably by the process of trans-splicing. It is of interest, evolutionarily and mechanistically, to determine the chemical structure of the 5' terminus of the precursor (medRNA) and product (mRNA). We demonstrate here that the cap structure of both is most probably 7-methylguanosine in a 5',5' triphosphate linkage, consistent with a precursor/product relationship.

  View details for Web of Science ID A1988L482800059

  View details for PubMedID 3336365

• PRECISE IDENTIFICATION OF CLEAVAGE SITES INVOLVED IN THE UNUSUAL PROCESSING OF TRYPANOSOME RIBOSOMAL-RNA JOURNAL OF MOLECULAR BIOLOGY Campbell, D. A., Kubo, K., Clark, C. G., Boothroyd, J. C. 1987; 196 (1): 113-124

  Abstract

  The large subunit ribosomal RNA (LSRNA) of Trypanosoma brucei is unusual in being cleaved at multiple sites to yield six stable fragments of RNA. We report here the complete nucleotide sequence of two regions of the ribosomal DNA repeat unit. The first sequence includes all of the processing sites involved in the generation of one of the small LSRNA fragments. The second region encodes the trypanosome 5.8 S RNA. By RNA sequencing and S1 nuclease mapping, we have identified the processing sites involved in the generation of both of these small RNAs. On the basis of predicted secondary structure models, we infer that all the cleavages apparently occur near the junction of single- and double-stranded regions. The sites involved in the novel LSRNA processing show a clear symmetry with respect to a conserved region of ten base-pairs. No such signals are evident for the processing sites that generate the 5.8 S RNA.

  View details for Web of Science ID A1987H996700009

  View details for PubMedID 3656442

• AN UNUSUALLY COMPACT RIBOSOMAL DNA REPEAT IN THE PROTOZOAN GIARDIA-LAMBLIA NUCLEIC ACIDS RESEARCH Boothroyd, J. C., Wang, A., Campbell, D. A., Wang, C. C. 1987; 15 (10): 4065-4084

  Abstract

  The ribosomal RNA (rRNA) genes of the protozoan parasite Giardia lamblia have been analyzed with respect to size, composition and copy number. They are found to be remarkable in several respects. First, the rRNAs themselves are the smallest yet reported for any eukaryotic organism. Second, the genes encoding them are found as an exceptionally small tandemly repeated unit of only 5.4 kilobase-pairs. Third, the genes are extraordinarily G:C rich, even in regions which are highly conserved between all other eukaryotic rRNA genes. Finally, by analogy to other organisms, the 5.8S RNA appears to lack about 15 nucleotides from its 3'-end, a region previously thought to be essential for 5.8S RNA function. We also provide the first estimates of the genomic complexity and total G:C content of this important protozoan pathogen.

  View details for Web of Science ID A1987H489600010

  View details for PubMedID 3588284

• EVIDENCE FOR TRANS SPLICING IN TRYPANOSOMES CELL Sutton, R. E., Boothroyd, J. C. 1986; 47 (4): 527-535

  Abstract

  The 5' ends of trypanosome mRNAs consist of an identical sequence of 35 nucleotides. This "mini-exon" sequence is derived from the 5' end of a 137 nucleotide RNA (medRNA). The remainder of each mRNA is derived from a protein-coding exon that is not linked to the mini-exon. We propose that medRNA is spliced in trans to de-novo-initiated transcripts of protein-coding genes. This trans splicing model predicts that the downstream portion of medRNA will be part of a branched structure and then be released as a free product (minRNA). We demonstrate that significant levels of minRNA exist in trypanosome RNA. Furthermore, minRNA can be released from high molecular weight RNA by a HeLa cell S100 "debranching" extract. We conclude that trans splicing is the physiological process by which mature mRNA molecules are synthesized in trypanosomes.

  View details for Web of Science ID A1986E984000007

  View details for PubMedID 3022935

• ANTIGENIC VARIATION IN AFRICAN TRYPANOSOMES ANNUAL REVIEW OF MICROBIOLOGY Boothroyd, J. C. 1985; 39: 475-502

  View details for Web of Science ID A1985ARS4100021

  View details for PubMedID 3904609

• APPARENT DISCONTINUOUS TRANSCRIPTION OF TRYPANOSOMA-BRUCEI VARIANT SURFACE-ANTIGEN GENES NATURE Campbell, D. A., THORNTON, D. A., Boothroyd, J. C. 1984; 311 (5984): 350-355

  Abstract

  The repeated mini-exon sequence that encodes the first 35 base pairs of all variant surface antigen mRNAs of Trypanosoma brucei directs the synthesis of a discrete 137-nucleotide transcript. It thus seems that variant surface antigen mRNAs are transcribed discontinuously, and we present two alternative models for how this might occur.

  View details for Web of Science ID A1984TK56000042

  View details for PubMedID 6090933

• THE 5'-LIMIT OF TRANSPOSITION AND UPSTREAM BARREN REGION OF A TRYPANOSOME VSG GENE - TANDEM-76 BASE-PAIR REPEATS FLANKING (TAA)90 NUCLEIC ACIDS RESEARCH Campbell, D. A., VANBREE, M. P., Boothroyd, J. C. 1984; 12 (6): 2759-2774

  Abstract

  We have cloned and sequenced a portion of the region upstream of an expressed VSG gene of Trypanosoma brucei. This "expression-linked copy" arose through the duplication and transposition of a silent, "basic copy" of the gene to an expression site. Comparison of the sequences surrounding the 5'-end of the transposed segment in the two loci indicates the 5'-limit of transposition lies within the first (3'-most) of three repeated segments found at this position in the basic copy locus. These highly conserved repeat segments which average 76 base-pairs in length are also found tandemly repeated upstream of the transposed segment in the expression site. In this latter site, however, they are more numerous (at least 17 repeats) and they are interrupted, within the middle of one repeat, by a 270 base-pair region consisting of (TAA)90. The possible roles of these unusual sequences in transposition and in a model proposing discontinuous transcription of VSG genes are discussed.

  View details for Web of Science ID A1984SK10500013

  View details for PubMedID 6324125

• COMPARISON OF THE AMINO-ACID-SEQUENCE OF THE MAJOR IMMUNOGEN FROM 3 SEROTYPES OF FOOT AND MOUTH-DISEASE VIRUS NUCLEIC ACIDS RESEARCH Makoff, A. J., PAYNTER, C. A., Rowlands, D. J., Boothroyd, J. C. 1982; 10 (24): 8285-8292

  Abstract

  Cloned cDNA molecules from three serotypes of FMDV have been sequenced around the VP1-coding region. The predicted amino acid sequences for VP1 were compared with the published sequences and variable regions identified. The amino acid sequences were also analysed for hydrophilic regions. Two of the variable regions, numbered 129-160 and 193-204 overlapped hydrophilic regions, and were therefore identified as potentially immunogenic. These regions overlap regions shown by others to be immunogenic.

  View details for Web of Science ID A1982PW32000029

  View details for PubMedID 6298715

• THE NUCLEOTIDE-SEQUENCE OF CDNA CODING FOR THE STRUCTURAL PROTEINS OF FOOT-AND-MOUTH-DISEASE VIRUS GENE Boothroyd, J. C., Harris, T. J., Rowlands, D. J., Lowe, P. A. 1982; 17 (2): 153-161

  Abstract

  The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.

  View details for Web of Science ID A1982NL19900006

  View details for PubMedID 6282711

• COMPLETE NUCLEOTIDE-SEQUENCE OF COMPLEMENTARY-DNA CODING FOR A VARIANT SURFACE GLYCOPROTEIN FROM TRYPANOSOMA-BRUCEI JOURNAL OF MOLECULAR BIOLOGY Boothroyd, J. C., PAYNTER, C. A., Coleman, S. L., Cross, G. A. 1982; 157 (3): 547-556

  View details for Web of Science ID A1982NT01300007

  View details for PubMedID 7120401

• TRANSCRIPTS CODING FOR VARIANT SURFACE GLYCOPROTEINS OF TRYPANOSOMA-BRUCEI HAVE A SHORT, IDENTICAL EXON AT THEIR 5' END GENE Boothroyd, J. C., Cross, G. A. 1982; 20 (2): 281-289

  View details for Web of Science ID A1982PY43800017

  View details for PubMedID 7166234

• MOLECULAR-CLONING OF FOOT AND MOUTH-DISEASE VIRUS GENOME AND NUCLEOTIDE-SEQUENCES IN THE STRUCTURAL PROTEIN GENES NATURE Boothroyd, J. C., HIGHFIELD, P. E., Cross, G. A., Rowlands, D. J., Lowe, P. A., Brown, F., Harris, T. J. 1981; 290 (5809): 800-802

  Abstract

  Foot and mouth disease virus (FMDV), of the family Picornaviridae, consists of a single-stranded RNA (approximately 8,000 nucleotides), the translation of which is initiated on the 3' side of a 150-nucleotide poly(C) tract and yields a single polyprotein which is processed by host cell proteases into four primary products (Fig. 1). One or more virus-specified proteases further cleave these into the final products, the capsid proteins (VP1-4) being derived from the precursor p88 (for review see ref. 5). There are seven serotypes of the virus and as it has been shown that the immunizing activity of FMDV particles is associated primarily with VP1 (refs 6, 7), it seems likely that antigenic variation in FMDV is a result of changes in the structure of this protein. To further our understanding of this variation and as a first step in the possible development of FMDV vaccines from genetically manipulated microorganisms, we report here the construction and analysis of recombinant plasmids containing cDNA copies of the RNA. Comparison of the deduced amino acid sequence with the known polypeptide sequences shows that the NH2-termini of VP2 and VP3 are conserved between the A and O serotypes whereas that of VP1 (the immunizing antigen) varies by as much as 42% between serotypes.

  View details for Web of Science ID A1981LM52000046

  View details for PubMedID 6261157

• VARIANT SURFACE GLYCOPROTEINS OF TRYPANOSOMA-BRUCEI ARE SYNTHESIZED WITH CLEAVABLE HYDROPHOBIC SEQUENCES AT THE CARBOXY AND AMINO TERMINI NUCLEIC ACIDS RESEARCH Boothroyd, J. C., PAYNTER, C. A., Cross, G. A., Bernards, A., BORST, P. 1981; 9 (18): 4735-4743

  Abstract

  cDNAs coding for the amino and carboxy termini of two trypanosome variant surface glycoproteins (VSGs) have been sequenced. The results indicate that VSGs are synthesised with hydrophobic amino-terminal leader and carboxy-terminal tail sequences which are absent from purified mature VSGs.

  View details for Web of Science ID A1981MK20600023

  View details for PubMedID 6272213

• ACTIVATION OF TRYPANOSOME SURFACE GLYCOPROTEIN GENES INVOLVES A DUPLICATION-TRANSPOSITION LEADING TO AN ALTERED 3' END CELL Bernards, A., VANDERPLOEG, L. H., FRASCH, A. C., BORST, P., Boothroyd, J. C., Coleman, S., Cross, G. A. 1981; 27 (3): 497-505

  Abstract

  Expression of the genes for variant surface glycoproteins 117 and 118 in Trypanosoma brucei is accompanied by the appearance of an extra copy of these genes, the expression-linked copy, which differs in the surrounding restriction enzyme sites from the corresponding basic copy of the genes. We present direct evidence that the expression-linked copy is the one used for messenger RNA synthesis. By S1-nuclease-protection experiments we show that cloned basic-copy genes contain the nucleotide sequence of the corresponding messenger RNA except for the last 100 to 150 nucleotides before the poly(A) tail. Comparison of the 3'-terminal sequence of the 117 basic-copy gene and the 117 complementary DNA shows that this region differs by multiple point mutations, insertions and deletions, the differences starting within the coding sequence. Genomic blots demonstrate that a Bsp I site in the 3'-terminal part of the 118 complementary DNA is present in the expression-linked copy but not in the basic-copy gene. We conclude that expression-linked copies are the active genes, and that the generation of expression-linked copies involves a duplication--transposition in which the 3' end of the gene is replaced.

  View details for Web of Science ID A1981MW20000011

  View details for PubMedID 6101223

• HOMOLOGOUS 3'-TERMINAL REGIONS OF MESSENGER-RNAS FOR SURFACE-ANTIGENS OF DIFFERENT ANTIGENIC VARIANTS OF TRYPANOSOMA-BRUCEI NUCLEIC ACIDS RESEARCH Majumder, H. K., Boothroyd, J. C., Weber, H. 1981; 9 (18): 4745-4753

  Abstract

  Sequences corresponding to the complete 3'-terminal regions of the messenger RNAs for three different Variant Surface Glycoproteins of Trypanosoma brucei were determined on complementary DNA inserts cloned in recombinant plasmids. The three sequences show 80-130 base pair long segments of strong (70-80%) homology at the 3' ends, whereas ther regions upstream from the last 130-140 base pairs contain no significant homology. The signal AAUAAA, present near the 3' ends of almost all known polyadenylated mRNAs of eukaryotes, does not occur in the 3'-terminal sequences of these three variants.

  View details for Web of Science ID A1981MK20600024

  View details for PubMedID 6170939

• AN INTRODUCTION TO ANTIGENIC VARIATION IN TRYPANOSOMES AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE Cross, G. A., Holder, A. A., Allen, G., Boothroyd, J. C. 1980; 29 (5): 1027-1032

  Abstract

  The salivarian trypanosomes have a unique capacity for antigenic variation at the cell surface. This phenomenon is their primary mechanism for evasion of the host's immune response. Variation is mediated through alternate expression of an extensive repertoire of variant surface glycoproteins (VSGs). Extensive amino acid sequence diversity is responsible for the antigenic diversity of VSGs. All the isolated VSGs of Trypanosoma brucei studied also contain an immunologically cross-reacting glycosyl side chain at the C-terminus, which probably represents a recognition site for proteolytic processing of the hydrophobic putative membrane-binding tail present on the synthesized molecule but not so far found on purified VSGs.

  View details for Web of Science ID A1980KW12700002

  View details for PubMedID 7435800

• A VARIANT SURFACE GLYCOPROTEIN OF TRYPANOSOMA-BRUCEI SYNTHESIZED WITH A C-TERMINAL HYDROPHOBIC TAIL ABSENT FROM PURIFIED GLYCOPROTEIN NATURE Boothroyd, J. C., Cross, G. A., Hoeijmakers, J. H., BORST, P. 1980; 288 (5791): 624-626

  Abstract

  Sequential expression of variant surface glycoproteins (VSGs) enables the parasitic protozoan Trypanosoma brucei to evade the immune response of its mammalian hosts. Studies of several VSGs, which have been isolated as soluble molecules following disruption of cells in the absence of detergent, have indicated extensive amino acid diversity and the absence of a hydrophobic segment which might serve to anchor the carboxy terminus to the membrane. The carboxy-terminal tryptic peptides of six VSGs have recently been characterized and shown to be glycosylated. Three of these VSGs terminated with a glycosylated aspartate or asparagine residue (Asx), suggesting that the VSG was cleaved following synthesis and glycosylation and before characterization. We present here nucleotide sequence data which suggest that the primary translation product of one VSG gene contains a hydrophobic tail at the carboxy terminus which is not found on the isolated, mature glycoprotein. The data also predict that the glycosylated residue is aspartic acid rather than the anticipated asparagine.

  View details for Web of Science ID A1980KT80300057

  View details for PubMedID 7442811

• NEW GENES AND PROMOTERS SUGGESTED BY THE DNA-SEQUENCE NEAR THE END OF THE COLIPHAGE-T7 EARLY OPERON NUCLEIC ACIDS RESEARCH Boothroyd, J. C., Hayward, R. S. 1979; 7 (7): 1931-1943

  Abstract

  We have employed the dideoxynucleotide chain-terminating method to determine the nucleotide sequence of T7 DNA between the physical map positions 18.9% and 19.8%. The most striking features of this sequence are two perfect 21-basepair repeats, each of which appears to contain a promoter for late transcription. In each case the promoter sequence incorporates a putative translational terminator on its left (5'-side of the "sense" strand), and overlaps a potential ribosome-binding site on its right. The region probably lies immediately distal to the early operon, and may contain two short, hitherto unreported protein-coding sequences.

  View details for Web of Science ID A1979HY88300013

  View details for PubMedID 231766

• SYNCHRONOUS GROWTH AND PLASTID REPLICATION IN NATURALLY WALL-LESS ALGA OLISTHODISCUS-LUTEUS PLANT PHYSIOLOGY Cattolico, R. A., Boothroyd, J. C., Gibbs, S. P. 1976; 57 (4): 497-503

  Abstract

  Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B(12) to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B(12), plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.

  View details for Web of Science ID A1976BP61300009

  View details for PubMedID 16659514