Honors & Awards
Medical Scientist Training Program (MSTP), NIH (2001-2008)
Career Award for Medical Scientists, Burroughs-Wellcome Fund (2012-2017)
Early Career Independence Award (DP5), NIH (2012-2017)
MD, Harvard Medical School, Medicine (2008)
PhD, Harvard Medical School, Biophysics (2006)
BA, Harvard University, Biochemical Sciences (2001)
Current Research and Scholarly Interests
Lab website: http://yehlab.stanford.edu/
The chemistry and biology of the unique plastid organelle, the apicoplast, in malaria parasites
Malaria caused by Plasmodium spp parasites has an enormous disease burden that disproportionately affects the worlds poorest and youngest. New anti-malarials with novel drug mechanisms are desperately needed in the face of existing or emerging drug resistance to all available therapies. Investigation of Plasmodium biology offers both the potential for important biomedical impact and an opportunity to explore fascinating eukaryotic biology. Given the challenges of genetic and other approaches to studying this complex organism, the development of chemical tools will be especially critical in pushing forward basic research.
My research focuses on the apicoplast, a prokaryotically-derived plastid organelle unique to Plasmodium (and other pathogenic Apicomplexa parasites) and a key anti-malarial drug target. My laboratory's goal is to elucidate apicoplast biology, function, and role in pathogenesis with the ultimate goal of realizing the potential of the apicoplast as a therapeutic target. In a major step toward this goal, my previous work has demonstrated that the sole essential function of the apicoplast in blood-stage P. falciparum parasites is the biosynthesis of isoprenoid precursors. As such, I was able to generate parasites completely devoid of this essential organelle but chemically rescued by supplementation of the growth media with isopentenyl pyrophosphate (IPP), the pathway product. Chemical rescue and apicoplast(-) parasites are innovative tools for investigating apicoplast biology and for advancing apicoplast-directed drug and vaccine development. Our research takes advantage of these new tools and our newfound understanding of apicoplast function to explore a variety of topics, including protein trafficking to the apicoplast and the protein "prenylome" in Plasmodium. We employ a variety of methods but have a particular focus on the use of chemical tools to overcome the current challenges in studying this organelle. Our exploration of the Plasmodium apicoplast are likely to reveal both unique biology and targets for anti-malarial drug development.
- Physician Scientist Hour
INDE 217 (Aut)
Independent Studies (11)
- Directed Reading in Biochemistry
BIOC 299 (Aut, Win, Spr, Sum)
- Directed Reading in Microbiology and Immunology
MI 299 (Win, Spr)
- Graduate Research
MI 399 (Aut, Win, Spr)
- Graduate Research
PATH 399 (Aut)
- Graduate Research and Special Advanced Work
BIOC 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
BIOC 370 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MI 370 (Win, Spr)
- The Teaching of Biochemistry
BIOC 221 (Aut, Win, Spr, Sum)
- Undergraduate Research
BIOC 199 (Aut, Win, Spr, Sum)
- Undergraduate Research
MI 199 (Win, Spr)
- Undergraduate Research
PATH 199 (Aut, Sum)
- Directed Reading in Biochemistry
CaaX-Like Protease of Cyanobacterial Origin Is Required for Complex Plastid Biogenesis in Malaria Parasites.
2020; 11 (5)
Plasmodium parasites and related apicomplexans contain an essential "complex plastid" organelle of secondary endosymbiotic origin, the apicoplast. Biogenesis of this complex plastid poses a unique challenge requiring evolution of new cellular machinery. We previously conducted a mutagenesis screen for essential apicoplast biogenesis genes to discover organellar pathways with evolutionary and biomedical significance. Here we validate and characterize a gene candidate from our screen, Pf3D7_0913500. Using a conditional knockdown strain, we show that Pf3D7_0913500 depletion causes growth inhibition that is rescued by the sole essential product of the apicoplast, isopentenyl pyrophosphate (IPP), and results in apicoplast loss. Because Pf3D7_0913500 had no previous functional annotation, we name it apicoplast-minus IPP-rescued 4 (AMR4). AMR4 has an annotated CaaX protease and bacteriocin processing (CPBP) domain, which in eukaryotes typically indicates a role in CaaX postprenylation processing. Indeed, AMR4 is the only putative CaaX-like protease in Plasmodium parasites which are known to require protein prenylation, and we confirm that the conserved catalytic residue of AMR4 (E352) is required for its apicoplast function. However, we unexpectedly find that AMR4 does not act in a CaaX postprenylation processing pathway in Plasmodium falciparum Instead, we find that AMR4 is imported into the apicoplast and is derived from a cyanobacterial CPBP gene which was retained through both primary and secondary endosymbiosis. Our findings suggest that AMR4 is not a true CaaX protease, but instead it performs a conserved, uncharacterized chloroplast function that has been retained for complex plastid biogenesis.IMPORTANCE Plasmodium parasites, which cause malaria, and related apicomplexans are important human and veterinary pathogens. These parasites represent a highly divergent and understudied branch of eukaryotes, and as such often defy the expectations set by model organisms. One striking example of unique apicomplexan biology is the apicoplast, an essential but nonphotosynthetic plastid derived from an unusual secondary (eukaryote-eukaryote) endosymbiosis. Endosymbioses are a major driver of cellular innovation, and apicoplast biogenesis pathways represent a hot spot for molecular evolution. We previously conducted an unbiased screen for apicoplast biogenesis genes in P. falciparum to uncover these essential and innovative pathways. Here, we validate a novel gene candidate from our screen and show that its role in apicoplast biogenesis does not match its functional annotation predicted by model eukaryotes. Our findings suggest that an uncharacterized chloroplast maintenance pathway has been reused for complex plastid biogenesis in this divergent branch of pathogens.
View details for DOI 10.1128/mBio.01492-20
View details for PubMedID 33024034
- Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity PLOS NEGLECTED TROPICAL DISEASES 2020; 14 (3)
Identification of anisomycin, prodigiosin and obatoclax as compounds with broad-spectrum anti-parasitic activity.
PLoS neglected tropical diseases
2020; 14 (3): e0008150
Parasitic infections are a major source of human suffering, mortality, and economic loss, but drug development for these diseases has been stymied by the significant expense involved in bringing a drug though clinical trials and to market. Identification of single compounds active against multiple parasitic pathogens could improve the economic incentives for drug development as well as simplifying treatment regimens. We recently performed a screen of repurposed compounds against the protozoan parasite Entamoeba histolytica, causative agent of amebic dysentery, and identified four compounds (anisomycin, prodigiosin, obatoclax and nithiamide) with low micromolar potency and drug-like properties. Here, we extend our investigation of these drugs. We assayed the speed of killing of E. histolytica trophozoites and found that all four have more rapid action than the current drug of choice, metronidazole. We further established a multi-institute collaboration to determine whether these compounds may have efficacy against other parasites and opportunistic pathogens. We found that anisomycin, prodigiosin and obatoclax all have broad-spectrum antiparasitic activity in vitro, including activity against schistosomes, T. brucei, and apicomplexan parasites. In several cases, the drugs were found to have significant improvements over existing drugs. For instance, both obatoclax and prodigiosin were more efficacious at inhibiting the juvenile form of Schistosoma than the current standard of care, praziquantel. Additionally, low micromolar potencies were observed against pathogenic free-living amebae (Naegleria fowleri, Balamuthia mandrillaris and Acanthamoeba castellanii), which cause CNS infection and for which there are currently no reliable treatments. These results, combined with the previous human use of three of these drugs (obatoclax, anisomycin and nithiamide), support the idea that these compounds could serve as the basis for the development of broad-spectrum anti-parasitic drugs.
View details for DOI 10.1371/journal.pntd.0008150
View details for PubMedID 32196500
A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites.
2019; 17 (2): e3000136
Endosymbiosis has driven major molecular and cellular innovations. Plasmodium spp. parasites that cause malaria contain an essential, non-photosynthetic plastid-the apicoplast-which originated from a secondary (eukaryote-eukaryote) endosymbiosis. To discover organellar pathways with evolutionary and biomedical significance, we performed a mutagenesis screen for essential genes required for apicoplast biogenesis in Plasmodium falciparum. Apicoplast(-) mutants were isolated using a chemical rescue that permits conditional disruption of the apicoplast and a new fluorescent reporter for organelle loss. Five candidate genes were validated (out of 12 identified), including a triosephosphate isomerase (TIM)-barrel protein that likely derived from a core metabolic enzyme but evolved a new activity. Our results demonstrate, to our knowledge, the first forward genetic screen to assign essential cellular functions to unannotated P. falciparum genes. A putative TIM-barrel enzyme and other newly identified apicoplast biogenesis proteins open opportunities to discover new mechanisms of organelle biogenesis, molecular evolution underlying eukaryotic diversity, and drug targets against multiple parasitic diseases.
View details for PubMedID 30726238
- Host Cell Metabolism Contributes to Delayed-Death Kinetics of Apicoplast Inhibitors in Toxoplasma gondii ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 2019; 63 (2)
- A mutagenesis screen for essential plastid biogenesis genes in human malaria parasites PLOS BIOLOGY 2019; 17 (2)
Disruption of Apicoplast Biogenesis by Chemical Stabilization of an Imported Protein Evades the Delayed-Death Phenotype in Malaria Parasites.
2019; 4 (1)
Malaria parasites (Plasmodium spp.) contain a nonphotosynthetic plastid organelle called the apicoplast, which houses essential metabolic pathways and is required throughout the parasite life cycle. The biogenesis pathways responsible for apicoplast growth, division, and inheritance are of key interest as potential drug targets. Unfortunately, several known apicoplast biogenesis inhibitors are of limited clinical utility because they cause a peculiar "delayed-death" phenotype in which parasites do not stop replicating until the second lytic cycle posttreatment. Identifying apicoplast biogenesis pathways that avoid the delayed-death phenomenon is a priority. Here, we generated parasites targeting a murine dihydrofolate reductase (mDHFR) domain, which can be conditionally stabilized with the compound WR99210, to the apicoplast. Surprisingly, chemical stabilization of this exogenous fusion protein disrupted parasite growth in an apicoplast-specific manner after a single lytic cycle. WR99210-treated parasites exhibited an apicoplast biogenesis defect beginning within the same lytic cycle as drug treatment, indicating that stabilized mDHFR perturbs a non-delayed-death biogenesis pathway. While the precise mechanism-of-action of the stabilized fusion is still unclear, we hypothesize that it inhibits apicoplast protein import by stalling within and blocking translocons in the apicoplast membranes.IMPORTANCE Malaria is a major cause of global childhood mortality. To sustain progress in disease control made in the last decade, new antimalarial therapies are needed to combat emerging drug resistance. Malaria parasites contain a relict chloroplast called the apicoplast, which harbors new targets for drug discovery. Unfortunately, some drugs targeting apicoplast pathways exhibit a delayed-death phenotype, which results in a slow onset-of-action that precludes their use as fast-acting, frontline therapies. Identification of druggable apicoplast biogenesis factors that will avoid the delayed-death phenotype is an important priority. Here, we find that chemical stabilization of an apicoplast-targeted mDHFR domain disrupts apicoplast biogenesis and inhibits parasite growth after a single lytic cycle, suggesting a non-delayed-death target. Our finding indicates that further interrogation of the mechanism-of-action of this exogenous fusion protein may reveal novel therapeutic avenues.
View details for PubMedID 30674649
- Disruption of Apicoplast Biogenesis by Chemical Stabilization of an Imported Protein Evades the Delayed-Death Phenotype in Malaria Parasites MSPHERE 2019; 4 (1)
- Plastid-endomembrane connections in apicomplexan parasites. PLoS pathogens 2019; 15 (6): e1007661
Host cell metabolism contributes to delayed-death kinetics of apicoplast inhibitors in Toxoplasma gondii.
Antimicrobial agents and chemotherapy
Toxoplasma gondii and related human parasites contain an essential plastid organelle called the apicoplast. Clinically-used antibiotics and other inhibitors that disrupt apicoplast biogenesis cause a mysterious "delayed-death" phenotype in which parasite growth is unaffected during the first lytic cycle of inhibitor treatment but is severely inhibited in the second lytic cycle even after drug removal. Critical to understanding the complex downstream cellular effects of these drug classes is the timing of apicoplast loss during inhibitor treatment and how it relates to this peculiar growth phenotype. Here we show that, upon treatment with diverse classes of apicoplast inhibitors, newly-replicated T. gondii parasites in the first lytic cycle initially form apicoplasts with defects in protein import or genome replication and eventually fail to inherit the apicoplast altogether. Despite the accumulation of parasites with defective or missing apicoplasts, growth is unaffected during the first lytic cycle, as previously observed. Strikingly, concomitant inhibition of host cell isoprenoid biosynthesis results in growth inhibition in the first lytic cycle and unmasks the apicoplast defects. These results suggest that defects in and even complete loss of the apicoplast in T. gondii are partially rescued by scavenging of host cell metabolites leading to death that is delayed. Our findings uncover host cell interactions that can alleviate apicoplast inhibition and highlight key differences in "delayed-death" inhibitors between T. gondii and Plasmodium falciparum.
View details for PubMedID 30455243
The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture.
2018; 3 (5)
Hydrolase are enzymes that regulate diverse biological processes, including posttranslational protein modifications. Recent work identified four active serine hydrolases (ASHs) in Toxoplasma gondii as candidate depalmitoylases. However, only TgPPT1 (ASH1) has been confirmed to remove palmitate from proteins. ASH4 (TgME49_264290) was reported to be refractory to genetic disruption. We demonstrate that recombinant ASH4 is an esterase that processes short acyl esters but not palmitoyl thioesters. Genetic disruption of ASH4 causes defects in cell division and premature scission of parasites from residual bodies. These defects lead to the presence of vacuoles with a disordered intravacuolar architecture, with parasites arranged in pairs around multiple residual bodies. Importantly, we found that the deletion of ASH4 correlates with a defect in radial dispersion from host cells after egress. This defect in dispersion of parasites is a general phenomenon that is observed for disordered vacuoles that occur at low frequency in wild-type parasites, suggesting a possible general link between intravacuolar organization and dispersion after egress.IMPORTANCE This work defines the function of an enzyme in the obligate intracellular parasite Toxoplasma gondii We show that this previously uncharacterized enzyme is critical for aspects of cellular division by the parasite and that loss of this enzyme leads to parasites with cell division defects and which also are disorganized inside their vacuoles. This leads to defects in the ability of the parasite to disseminate from the site of an infection and may have a significant impact on the parasite's overall infectivity of a host organism.
View details for PubMedID 30232166
Integrative proteomics and bioinformatic prediction enable a high-confidence apicoplast proteome in malaria parasites.
2018; 16 (9): e2005895
Malaria parasites (Plasmodium spp.) and related apicomplexan pathogens contain a nonphotosynthetic plastid called the apicoplast. Derived from an unusual secondary eukaryote-eukaryote endosymbiosis, the apicoplast is a fascinating organelle whose function and biogenesis rely on a complex amalgamation of bacterial and algal pathways. Because these pathways are distinct from the human host, the apicoplast is an excellent source of novel antimalarial targets. Despite its biomedical importance and evolutionary significance, the absence of a reliable apicoplast proteome has limited most studies to the handful of pathways identified by homology to bacteria or primary chloroplasts, precluding our ability to study the most novel apicoplast pathways. Here, we combine proximity biotinylation-based proteomics (BioID) and a new machine learning algorithm to generate a high-confidence apicoplast proteome consisting of 346 proteins. Critically, the high accuracy of this proteome significantly outperforms previous prediction-based methods and extends beyond other BioID studies of unique parasite compartments. Half of identified proteins have unknown function, and 77% are predicted to be important for normal blood-stage growth. We validate the apicoplast localization of a subset of novel proteins and show that an ATP-binding cassette protein ABCF1 is essential for blood-stage survival and plays a previously unknown role in apicoplast biogenesis. These findings indicate critical organellar functions for newly discovered apicoplast proteins. The apicoplast proteome will be an important resource for elucidating unique pathways derived from secondary endosymbiosis and prioritizing antimalarial drug targets.
View details for PubMedID 30212465
- The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture MSPHERE 2018; 3 (5)
The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture (vol 3, e00393-18, 2018)
2018; 3 (5)
View details for Web of Science ID 000449522900054
- The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture (vol 3, e00393-18, 2018) MSPHERE 2018; 3 (5)
Specific Inhibition of the Bifunctional Farnesyl/Geranylgeranyl Diphosphate Synthase in Malaria Parasites via a New Small-Molecule Binding Site
CELL CHEMICAL BIOLOGY
2018; 25 (2): 185-+
The bifunctional farnesyl/geranylgeranyl diphosphate synthase (FPPS/GGPPS) is a key branchpoint enzyme in isoprenoid biosynthesis in Plasmodium falciparum (malaria) parasites. PfFPPS/GGPPS is a validated, high-priority antimalarial drug target. Unfortunately, current bisphosphonate drugs that inhibit FPPS and GGPPS enzymes by acting as a diphosphate substrate analog show poor bioavailability and selectivity for PfFPPS/GGPPS. We identified a new non-bisphosphonate compound, MMV019313, which is highly selective for PfFPPS/GGPPS and showed no activity against human FPPS or GGPPS. Inhibition of PfFPPS/GGPPS by MMV019313, but not bisphosphonates, was disrupted in an S228T variant, demonstrating that MMV019313 and bisphosphonates have distinct modes of inhibition. Molecular docking indicated that MMV019313 did not bind previously characterized substrate sites in PfFPPS/GGPPS. Our finding uncovers a new, selective small-molecule binding site in this important antimalarial drug target with superior druggability compared with the known inhibitor site and sets the stage for the development of Plasmodium-specific FPPS/GGPPS inhibitors.
View details for PubMedID 29276048
ATG8 Is Essential Specifically for an Autophagy-Independent Function in Apicoplast Biogenesis in Blood-Stage Malaria Parasites.
2018; 9 (1)
Plasmodium parasites and related pathogens contain an essential nonphotosynthetic plastid organelle, the apicoplast, derived from secondary endosymbiosis. Intriguingly, a highly conserved eukaryotic protein, autophagy-related protein 8 (ATG8), has an autophagy-independent function in the apicoplast. Little is known about the novel apicoplast function of ATG8 and its importance in blood-stage Plasmodiumfalciparum Using a P.falciparum strain in which ATG8 expression was conditionally regulated, we showed that P. falciparum ATG8 (PfATG8) is essential for parasite replication. Significantly, growth inhibition caused by the loss of PfATG8 was reversed by addition of isopentenyl pyrophosphate (IPP), which was previously shown to rescue apicoplast defects in P.falciparum Parasites deficient in PfATG8, but whose growth was rescued by IPP, had lost their apicoplast. We designed a suite of functional assays, including a new fluorescence in situ hybridization (FISH) method for detection of the low-copy-number apicoplast genome, to interrogate specific steps in apicoplast biogenesis and detect apicoplast defects which preceded the block in parasite replication. Though protein import and membrane expansion of the apicoplast were unaffected, the apicoplast was not inherited by daughter parasites. Our findings demonstrate that, though multiple autophagy-dependent and independent functions have been proposed for PfATG8, only its role in apicoplast biogenesis is essential in blood-stage parasites. We propose that PfATG8 is required for fission or segregation of the apicoplast during parasite replication.IMPORTANCEPlasmodium parasites, which cause malaria, and related apicomplexan parasites are important human and veterinary pathogens. They are evolutionarily distant from traditional model organisms and possess a unique plastid organelle, the apicoplast, acquired by an unusual eukaryote-eukaryote endosymbiosis which established novel protein/lipid import and organelle inheritance pathways in the parasite cell. Though the apicoplast is essential for parasite survival in all stages of its life cycle, little is known about these novel biogenesis pathways. We show that malaria parasites have adapted a highly conserved protein required for macroautophagy in yeast and mammals to function specifically in apicoplast inheritance. Our finding elucidates a novel mechanism of organelle biogenesis, essential for pathogenesis, in this divergent branch of pathogenic eukaryotes.
View details for DOI 10.1128/mBio.02021-17
View details for PubMedID 29295911
- Erratum for Foe et al., "The Toxoplasma gondii Active Serine Hydrolase 4 Regulates Parasite Division and Intravacuolar Parasite Architecture". mSphere 2018; 3 (5)
Small molecule inhibition of apicomplexan FtsH1 disrupts plastid biogenesis in human pathogens
The malaria parasite Plasmodium falciparum and related apicomplexan pathogens contain an essential plastid organelle, the apicoplast, which is a key anti-parasitic target. Derived from secondary endosymbiosis, the apicoplast depends on novel, but largely cryptic, mechanisms for protein/lipid import and organelle inheritance during parasite replication. These critical biogenesis pathways present untapped opportunities to discover new parasite-specific drug targets. We used an innovative screen to identify actinonin as having a novel mechanism-of-action inhibiting apicoplast biogenesis. Resistant mutation, chemical-genetic interaction, and biochemical inhibition demonstrate that the unexpected target of actinonin in P. falciparum and Toxoplasma gondii is FtsH1, a homolog of a bacterial membrane AAA+ metalloprotease. PfFtsH1 is the first novel factor required for apicoplast biogenesis identified in a phenotypic screen. Our findings demonstrate that FtsH1 is a novel and, importantly, druggable antimalarial target. Development of FtsH1 inhibitors will have significant advantages with improved drug kinetics and multistage efficacy against multiple human parasites.
View details for DOI 10.7554/eLife.29865
View details for Web of Science ID 000408633800001
View details for PubMedID 28826494
View details for PubMedCentralID PMC5576918
The Prenylated Proteome of Plasmodium falciparum Reveals Pathogen-specific Prenylation Activity and Drug Mechanism-of-action
MOLECULAR & CELLULAR PROTEOMICS
2017; 16 (4): S54-S64
Plasmodium parasites contain several unique membrane compartments in which prenylated proteins may play important roles in pathogenesis. Protein prenylation has also been proposed as an antimalarial drug target because farnesyltransferase inhibitors cause potent growth inhibition of blood-stage Plasmodium However, the specific prenylated proteins that mediate antimalarial activity have yet to be identified. Given the potential for new parasite biology and elucidating drug mechanism-of-action, we performed a large-scale identification of the prenylated proteome in blood-stage P. falciparum parasites using an alkyne-labeled prenyl analog to specifically enrich parasite prenylated proteins. Twenty high-confidence candidates were identified, including several examples of pathogen-specific prenylation activity. One unique parasite prenylated protein was FYVE-containing coiled-coil protein (FCP), which is only conserved in Plasmodium and related Apicomplexan parasites and localizes to the parasite food vacuole. Targeting of FCP to this parasite-specific compartment was dependent on prenylation of its CaaX motif, as mutation of the prenylation site caused cytosolic mislocalization. We also showed that PfRab5b, which lacks C-terminal cysteines that are the only known site of Rab GTPase modification, is prenylated. Finally, we show that the THQ class of farnesyltransferase inhibitors abolishes FCP prenylation and causes its mislocalization, providing the first demonstration of a specific prenylated protein disrupted by antimalarial farnesyl transferase inhibitors. Altogether, these findings identify prenylated proteins that reveal unique parasite biology and are useful for evaluating prenyltransferase inhibitors for antimalarial drug development.
View details for DOI 10.1074/mcp.M116.064550
View details for Web of Science ID 000398812800006
View details for PubMedCentralID PMC5393391
The apicoplast: now you see it, now you don't
INTERNATIONAL JOURNAL FOR PARASITOLOGY
2017; 47 (2-3): 137-144
Parasites such as Plasmodium and Toxoplasma possess a vestigial plastid homologous to the chloroplasts of algae and plants. The plastid (known as the apicoplast; for apicomplexan plastid) is non-photosynthetic and very much reduced, but has clear endosymbiotic ancestry including a circular genome that encodes RNAs and proteins and a suite of bacterial biosynthetic pathways. Here we review the initial discovery of the apicoplast, and recount the major new insights into apicoplast origin, biogenesis and function. We conclude by examining how the apicoplast can be removed from malaria parasites in vitro, ultimately completing its reduction by chemical supplementation.
View details for DOI 10.1016/j.ijpara.2016.08.005
View details for Web of Science ID 000394925600009
A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis.
Antimicrobial agents and chemotherapy
2015; 59 (1): 356-364
The apicoplast is an essential plastid organelle found in Plasmodium parasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the "Malaria Box" library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stage Plasmodium falciparum growth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations in P. falciparum IspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activity in vitro with a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.
View details for DOI 10.1128/AAC.03342-14
View details for PubMedID 25367906
Chemical Rescue of Malaria Parasites Lacking an Apicoplast Defines Organelle Function in Blood-Stage Plasmodium falciparum
2011; 9 (8)
Plasmodium spp parasites harbor an unusual plastid organelle called the apicoplast. Due to its prokaryotic origin and essential function, the apicoplast is a key target for development of new anti-malarials. Over 500 proteins are predicted to localize to this organelle and several prokaryotic biochemical pathways have been annotated, yet the essential role of the apicoplast during human infection remains a mystery. Previous work showed that treatment with fosmidomycin, an inhibitor of non-mevalonate isoprenoid precursor biosynthesis in the apicoplast, inhibits the growth of blood-stage P. falciparum. Herein, we demonstrate that fosmidomycin inhibition can be chemically rescued by supplementation with isopentenyl pyrophosphate (IPP), the pathway product. Surprisingly, IPP supplementation also completely reverses death following treatment with antibiotics that cause loss of the apicoplast. We show that antibiotic-treated parasites rescued with IPP over multiple cycles specifically lose their apicoplast genome and fail to process or localize organelle proteins, rendering them functionally apicoplast-minus. Despite the loss of this essential organelle, these apicoplast-minus auxotrophs can be grown indefinitely in asexual blood stage culture but are entirely dependent on exogenous IPP for survival. These findings indicate that isoprenoid precursor biosynthesis is the only essential function of the apicoplast during blood-stage growth. Moreover, apicoplast-minus P. falciparum strains will be a powerful tool for further investigation of apicoplast biology as well as drug and vaccine development.
View details for DOI 10.1371/journal.pbio.1001138
View details for Web of Science ID 000294483000019
View details for PubMedID 21912516
Immediate Incubation Reduces Indeterminate Results for QuantiFERON-TB Gold In-Tube Assay
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (8): 2672-2676
In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37 degrees C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.
View details for DOI 10.1128/JCM.00482-10
View details for PubMedID 20519472
Real-Time PCR Testing for mecA Reduces Vancomycin Usage and Length of Hospitalization for Patients Infected with Methicillin-Sensitive Staphylococci
JOURNAL OF CLINICAL MICROBIOLOGY
2010; 48 (3): 785-790
Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to beta-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.
View details for DOI 10.1128/JCM.02150-09
View details for Web of Science ID 000274996200016
View details for PubMedID 20071556
View details for PubMedCentralID PMC2832423
Preferential Lower Respiratory Tract Infection in Swine-Origin 2009 A(H1N1) Influenza
CLINICAL INFECTIOUS DISEASES
2010; 50 (3): 391-394
We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.
View details for DOI 10.1086/649875
View details for Web of Science ID 000273500300014
View details for PubMedID 20047483
Hair Sheep Blood, Citrated or Defibrinated, Fulfills All Requirements of Blood Agar for Diagnostic Microbiology Laboratory Tests
2009; 4 (7)
Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.
View details for DOI 10.1371/journal.pone.0006141
View details for Web of Science ID 000267806300010
View details for PubMedID 19578541
View details for PubMedCentralID PMC2700971
Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases
2007; 46 (5): 1284-1292
The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.
View details for DOI 10.1021/bi0621213
View details for Web of Science ID 000243839500016
View details for PubMedID 17260957
Characterization of the aminocarboxycyclopropane-forming enzyme CmaC
2007; 46 (2): 359-368
The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a gamma-chloro-L-allo-Ile intermediate while covalently tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the gamma-Cl group by the alpha carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free gamma-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn-CmaC, acting on gamma-chloro-L-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the gamma-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a gamma-Cl-aminobutyryl alpha-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C-C bond formation and no azetidine carboxylate from an alternate N-C cyclization. CmaC could cyclize gamma,gamma-dichloroaminobutyryl to the Cl-ACC product but did not cyclize delta- or epsilon-chloroaminoacyl-S-CmaD substrates.
View details for DOI 10.1021/bi061930j
View details for Web of Science ID 000243337200004
View details for PubMedID 17209546
Enzymatic generation of the antimetabolite gamma,gamma-dichloroaminobutyrate by NRPS and mononuclear iron halogenase action in a streptomycete
CHEMISTRY & BIOLOGY
2006; 13 (11): 1183-1191
Four adjacent open reading frames, cytC1-C4, were cloned from a cytotrienin-producing strain of a Streptomyces sp. by using primers derived from the conserved region of a gene encoding a nonheme iron halogenase, CmaB, in coronamic acid biosynthesis. CytC1-3 were active after expression in Escherichia coli, and CytC4 was active after expression in Pseudomonas putida. CytC1, a relatively promiscuous adenylation enzyme, installs the aminoacyl moieties on the phosphopantetheinyl arm of the holo carrier protein CytC2. CytC3 is a nonheme iron halogenase that will generate both gamma-chloro- and gamma,gamma-dichloroaminobutyryl-S-CytC2 from aminobutyryl-S-CytC2. CytC4, a thioesterase, hydrolytically releases the dichloroaminobutyrate, a known streptomycete antibiotic. Thus, this short four-protein pathway is likely the biosynthetic source of this amino acid antimetabolite. This four-enzyme system analogously converts the proS-methyl group of valine to the dichloromethyl product regio- and stereospecifically.
View details for DOI 10.1016/j.chembiol.2006.09.012
View details for Web of Science ID 000242418400010
View details for PubMedID 17114000
- Nature's inventory of halogenation catalysts: Oxidative strategies predominate CHEMICAL REVIEWS 2006; 106 (8): 3364-3378
Flavin redox chemistry precedes substrate chlorination during the reaction of the flavin-dependent halogenase RebH
2006; 45 (25): 7904-7912
The flavin-dependent halogenase RebH catalyzes chlorination at the C7 position of tryptophan as the initial step in the biosynthesis of the chemotherapeutic agent rebeccamycin. The reaction requires reduced FADH(2) (provided by a partner flavin reductase), chloride ion, and oxygen as cosubstrates. Given the similarity of its sequence to those of flavoprotein monooxygenases and their common cosubstrate requirements, the reaction of FADH(2) and O(2) in the halogenase active site was presumed to form the typical FAD(C4a)-OOH intermediate observed in monooxygenase reactions. By using stopped-flow spectroscopy, formation of a FAD(C4a)-OOH intermediate was detected during the RebH reaction. This intermediate decayed to yield a FAD(C4a)-OH intermediate. The order of addition of FADH(2) and O(2) was critical for accumulation of the FAD(C4a)-OOH intermediate and for subsequent product formation, indicating that conformational dynamics may be important for protection of labile intermediates formed during the reaction. Formation of flavin intermediates did not require tryptophan, nor were their rates of formation affected by the presence of tryptophan, suggesting that tryptophan likely does not react directly with any flavin intermediates. Furthermore, although final oxidation to FAD occurred with a rate constant of 0.12 s(-)(1), quenched-flow kinetic data showed that the rate constant for 7-chlorotryptophan formation was 0.05 s(-)(1) at 25 degrees C. The kinetic analysis establishes that substrate chlorination occurs after completion of flavin redox reactions. These findings are consistent with a mechanism whereby hypochlorite is generated in the RebH active site from the reaction of FADH(2), chloride ion, and O(2).
View details for DOI 10.1021/bi060607d
View details for Web of Science ID 000238386300024
View details for PubMedID 16784243
Dichlorination of a pyrrolyl-S-carrier protein by FADH(2)-dependent halogenase PltA during pyoluteorin biosynthesis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (39): 13843-13848
The antifungal natural product pyoluteorin contains a 4,5-dichloropyrrole moiety. The timing of dichlorination in the heteroaromatic ring is now shown to occur after proline is tethered by thioester linkage to the carrier protein PltL and enzymatically desaturated to the pyrrolyl-S-PltL. Surprisingly, the FADH2-dependent halogenase PltA catalyzes chlorination at both positions of the ring, generating the 5-chloropyrrolyl-S-PltL intermediate and then the 4,5-dichloropyrrolyl-S-PltL product. PltA activity strictly depends on a heterologous flavin reductase that uses NAD(P)H to produce FADH2. Electrospray ionization-Fourier transform MS detected five covalent intermediates attached to the 11-kDa carrier protein PltL. Tandem MS localized the site of covalent modification on the carrier protein scaffold. HPLC analysis of the hydrolyzed products was consistent with the regiospecific chlorination at position 5 and then position 4 of the heteroaromatic ring. A mechanism for dichlorination is proposed involving formation of a FAD-4a-OCl intermediate for capture by the electron-rich C4 and C5 of the heteroaromatic pyrrole moiety.
View details for DOI 10.1073/pnas.0506964102
View details for Web of Science ID 000232231900026
View details for PubMedID 16162666
Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis
2005; 436 (7054): 1191-1194
Enzymatic incorporation of chlorine, bromine or iodine atoms occurs during the biosynthesis of more than 4,000 natural products. Halogenation can have significant consequences for the bioactivity of these products so there is great interest in understanding the biological catalysts that perform these reactions. Enzymes that halogenate unactivated aliphatic groups have not previously been characterized. Here we report the activity of five proteins-CmaA, CmaB, CmaC, CmaD and CmaE-in the construction of coronamic acid (CMA; 1-amino-1-carboxy-2-ethylcyclopropane), a constituent of the phytotoxin coronatine synthesized by the phytopathogenic bacterium Pseudomonas syringae. CMA derives from l-allo-isoleucine, which is covalently attached to CmaD through the actions of CmaA, a non-ribosomal peptide synthetase module, and CmaE, an unusual acyltransferase. We show that CmaB, a member of the non-haem Fe(2+), alpha-ketoglutarate-dependent enzyme superfamily, is the first of its class to show halogenase activity, chlorinating the gamma-position of l-allo-isoleucine. Another previously undescribed enzyme, CmaC, catalyses the formation of the cyclopropyl ring from the gamma-Cl-l-allo-isoleucine product of the CmaB reaction. Together, CmaB and CmaC execute gamma-halogenation followed by intramolecular gamma-elimination, in which biological chlorination is a cryptic strategy for cyclopropyl ring formation.
View details for DOI 10.1038/nature03797
View details for Web of Science ID 000231416600053
View details for PubMedID 16121186
Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2005; 102 (11): 3960-3965
The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH2 for the halogenase. Maximal rates were achieved at a RebF/RebH ratio of 3:1. In air-saturated solutions, a k(cat) of 1.4 min(-1) was observed for the RebF/RebH system but increased at least 10-fold in low-pO2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF/RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.
View details for DOI 10.1073/pnas.0500755102
View details for Web of Science ID 000227731000015
View details for PubMedID 15743914
Enhanced macrocyclizing activity of the thioesterase from tyrocidine synthetase in presence of nonionic detergent
CHEMISTRY & BIOLOGY
2004; 11 (11): 1573-1582
Macrocyclization carried out by thioesterase domains of multimodular nonribosomal peptide synthetases (NRPSs) is a key step in the biosynthesis of many biologically active peptides. The thioesterase excised from tyrocidine synthetase is a versatile macrocyclization catalyst and a useful tool for chemoenzymatic synthesis of diverse cyclic peptides. However, its utility is limited by its short lifetime of catalytic activity as well as significant flux of the acyl-enzyme intermediate to hydrolysis. The addition of Brij 58, a nonionic detergent, above the critical micelle concentration, has dramatic effects on enzyme activity: catalytic activity is extended to >60 min and the rate of cyclization (but not hydrolysis) increases 6-fold, resulting in a net 150- to 300-fold increase in cyclic product yields. This enhanced activity allowed enzymatic macrocyclization of a solid phase library of tyrocidine decapeptides to identify acceptable substitutions at the Orn9 position which had previously been inaccessible for diversification.
View details for DOI 10.1016/j.chembiol.2004.09.003
View details for Web of Science ID 000225613800016
View details for PubMedID 15556008
- Type II thioesterase restores activity of a NRPS module stalled with an aminoacyl-S-enzyme that cannot be elongated CHEMBIOCHEM 2004; 5 (9): 1290-1293