All Publications

  • Stress Response in Entamoeba histolytica Is Associated with Robust Processing of tRNA to tRNA Halves. mBio Sharma, M., Zhang, H., Ehrenkaufer, G., Singh, U. 2023: e0345022


    tRNA-derived fragments have been reported in many different organisms and have diverse cellular roles, such as regulating gene expression, inhibiting protein translation, silencing transposable elements, and modulating cell proliferation. In particular, tRNA halves, a class of tRNA fragments produced by the cleavage of tRNAs in the anti-codon loop, have been widely reported to accumulate under stress and regulate translation in cells. Here, we report the presence of tRNA-derived fragments in Entamoeba, with tRNA halves being the most abundant. We further established that tRNA halves accumulate in the parasites upon different stress stimuli such as oxidative stress, heat shock, and serum starvation. We also observed differential expression of tRNA halves during developmental changes of trophozoite-to-cyst conversion, with various tRNA halves accumulating during early encystation. In contrast to other systems, the stress response does not appear to be mediated by a few specific tRNA halves, as multiple tRNAs appear to be processed during the various stresses. Furthermore, we identified some tRNA-derived fragments associated with Entamoeba Argonaute proteins, EhAgo2-2 and EhAgo2-3, which have a preference for different tRNA-derived fragment species. Finally, we show that tRNA halves are packaged inside extracellular vesicles secreted by amoebas. The ubiquitous presence of tRNA-derived fragments, their association with the Argonaute proteins, and the accumulation of tRNA halves during multiple different stresses, including encystation, suggest a nuanced level of gene expression regulation mediated by different tRNA-derived fragments in Entamoeba. IMPORTANCE In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba. tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation. We also found that shorter tRNA-derived fragments are bound to Entamoeba Argonaute proteins, indicating that they may have a potential role in the Argonaute-mediated RNA-interference pathway, which mediates robust gene silencing in Entamoeba. We noticed that in response to heat shock, the protein translation levels were elevated in the parasites. This effect was reversed in the presence of an analog of leucine, which also reduced the levels of the tRNA halves in the stressed cells. Our results suggest that tRNA-derived fragments in Entamoeba have a possible role in regulating gene expression during environmental stress.

    View details for DOI 10.1128/mbio.03450-22

    View details for PubMedID 36809068

  • Characterization of extracellular vesicles from Entamoeba histolytica identifies roles in intercellular communication that regulates parasite growth and development. Infection and immunity Sharma, M. n., Morgado, P. n., Zhang, H. n., Ehrenkaufer, G. n., Manna, D. n., Singh, U. n. 2020


    Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins including proteins associated with vesicle formation, cell-signaling, metabolism, and cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27nt in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNAi pathway proteins, including Argonaute were also present in amebic EVs. Interestingly, we found that the amebic EVs impact inter-cellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveals that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication amongst parasites and influence growth kinetics.

    View details for DOI 10.1128/IAI.00349-20

    View details for PubMedID 32719158

  • Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets CELLULAR MICROBIOLOGY Sharma, M., Recuero-Checa, M. A., Fan, F., Dean, D. 2018; 20 (2)

    View details for DOI 10.1111/cmi.12801

    View details for Web of Science ID 000419773900003

  • Chlamydia trachomatis regulates growth and development in response to host cell fatty acid availability in the absence of lipid droplets. Cellular microbiology Sharma, M., Recuero-Checa, M. A., Fan, F. Y., Dean, D. 2018; 20 (2)


    Chlamydia trachomatis (Ct) is a Gram-negative obligate intracellular pathogen of humans that causes significant morbidity from sexually transmitted and ocular diseases globally. Ct acquires host fatty acids (FA) to meet the metabolic and growth requirements of the organism. Lipid droplets (LDs) are storehouses of FAs in host cells and have been proposed to be a source of FAs for the parasitophorous vacuole, termed inclusion, in which Ct replicates. Previously, cells devoid of LDs were shown to produce reduced infectious progeny at 24 hr postinfection (hpi). Here, although we also found reduced progeny at 24 hpi, there were significantly more progeny at 48 hpi in the absence of LDs compared to the control wild-type (WT) cells. These findings were confirmed using transmission electron microscopy where cells without LDs were shown to have significantly more metabolically active reticulate bodies at 24 hpi and significantly more infectious but metabolically inert elementary bodies at 48 hpi than WT cells. Furthermore, by measuring basal oxygen consumption rates (OCR) using extracellular flux analysis, Ct infected cells without LDs had higher OCRs at 24 hpi than cells with LDs, confirming ongoing metabolic activity in the absence of LDs. Although the FA oleic acid is a major source of phospholipids for Ct and stimulates LD synthesis, treatment with oleic acid, but not other FAs, enhanced growth and led to an increase in basal OCR in both LD depleted and WT cells, indicating that FA transport to the inclusion is not affected by the loss of LDs. Our results show that Ct regulates inclusion metabolic activity and growth in response to host FA availability in the absence of LDs.

    View details for DOI 10.1111/cmi.12801

    View details for PubMedID 29117636

    View details for PubMedCentralID PMC5764794

  • Chlamydia trachomatis growth and development requires the activity of host Long-chain Acyl-CoA Synthetases (ACSLs) SCIENTIFIC REPORTS Recuero-Checa, M. A., Sharma, M., Lau, C., Watkins, P. A., Gaydos, C. A., Dean, D. 2016; 6: 23148


    The obligate-intracellular pathogen Chlamydia trachomatis (Ct) has undergone considerable genome reduction with consequent dependence on host biosynthetic pathways, metabolites and enzymes. Long-chain acyl-CoA synthetases (ACSLs) are key host-cell enzymes that convert fatty acids (FA) into acyl-CoA for use in metabolic pathways. Here, we show that the complete host ACSL family [ACSL1 and ACSL3-6] translocates into the Ct membrane-bound vacuole, termed inclusion, and remains associated with membranes of metabolically active forms of Ct throughout development. We discovered that three different pharmacologic inhibitors of ACSL activity independently impede Ct growth in a dose-dependent fashion. Using an FA competition assay, host ACSLs were found to activate Ct branched-chain FAs, suggesting that one function of the ACSLs is to activate Ct FAs and host FAs (recruited from the cytoplasm) within the inclusion. Because the ACSL inhibitors can deplete lipid droplets (LD), we used a cell line where LD synthesis was switched off to evaluate whether LD deficiency affects Ct growth. In these cells, we found no effect on growth or on translocation of ACSLs into the inclusion. Our findings support an essential role for ACSL activation of host-cell and bacterial FAs within the inclusion to promote Ct growth and development, independent of LDs.

    View details for DOI 10.1038/srep23148

    View details for Web of Science ID 000372346200001

    View details for PubMedID 26988341

    View details for PubMedCentralID PMC4796813

  • A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells CELL REPORTS Jolly, A. L., Luan, C., Dusel, B. E., Dunne, S. F., Winding, M., Dixit, V. J., Robins, C., Saluk, J. L., Logan, D. J., Carpenter, A. E., Sharma, M., Dean, D., Cohen, A. R., Gelfand, V. I. 2016; 14 (3): 611–20


    Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

    View details for DOI 10.1016/j.celrep.2015.12.051

    View details for Web of Science ID 000368701600020

    View details for PubMedID 26774481

    View details for PubMedCentralID PMC4868501

  • HIF-1 alpha is involved in mediating apoptosis resistance to Chlamydia trachomatis-infected cells CELLULAR MICROBIOLOGY Sharma, M., Machuy, N., Boehme, L., Karunakaran, K., Maeurer, A. P., Meyer, T. F., Rudel, T. 2011; 13 (10): 1573–85


    Chlamydiae are obligate intracellular Gram-negative bacteria that cause widespread diseases in humans. Due to the intimate association between bacterium and host, Chlamydia evolved various strategies to protect their host cell against death-inducing stimuli, allowing the bacterium to complete its development cycle. An RNA interference (RNAi)-based screen was used to identify host cell factors required for apoptosis resistance of human epithelial cells infected with Chlamydia trachomatis serovar L2. Among the 32 validated hits, the anti-apoptotic Bcl-2 family member Mcl-1 was identified as a target. Protein network analyses implicated the transcription factor hypoxia-induced factor 1 alpha (HIF-1α) to be central to the regulation of many of the identified targets. Further mechanistic investigations showed that HIF-1α was stabilized within the host cell cytoplasm during early infection time points, followed by its translocation to the nucleus and eventual transcriptional activation of Mcl-1. siRNA-mediated depletion of HIF-1α led to a drastic decrease in Mcl-1, rendering the cell sensitive to apoptosis induction. Taken together, our findings identify HIF-1α as responsible for upregulation of Mcl-1 and the maintenance of apoptosis resistance during Chlamydia infection.

    View details for DOI 10.1111/j.1462-5822.2011.01642.x

    View details for Web of Science ID 000294924500011

    View details for PubMedID 21824245

  • Apoptosis resistance in Chlamydia-infected cells: a fate worse than death? FEMS IMMUNOLOGY AND MEDICAL MICROBIOLOGY Sharma, M., Rudel, T. 2009; 55 (2): 154–61


    Chlamydia has long been studied as an intracellular pathogen causing widespread diseases. In the last three decades, the field of apoptosis has rapidly emerged, and as a consequence, research on infectious diseases in general and on Chlamydia-host interaction in particular shifted to apoptosis modulation. Ten years ago, the first paper describing the drastic inhibition of apoptosis in Chlamydia-infected cells was published. In a reversal of roles, here was a pathogen that was strongly protecting cells in an organism against destruction by the organism's immune system. Since then, numerous studies have described apoptosis inhibition by Chlamydia and the mechanisms involved, but still there is a lack of general consensus on the subject. With a section of studies even reporting the induction of cell death by Chlamydia and not its inhibition, the field became even more diverse and complicated. In this review, an attempt is made to discuss the recent findings on apoptosis modulation by chlamydial species.

    View details for DOI 10.1111/j.1574-695X.2008.00515.x

    View details for Web of Science ID 000263133600006

    View details for PubMedID 19281566

  • Mcl-1 Is a Key Regulator of Apoptosis Resistance in Chlamydia trachomatis-Infected Cells PLOS ONE Rajalingam, K., Sharma, M., Lohmann, C., Oswald, M., Thieck, O., Froelich, C. J., Rudel, T. 2008; 3 (9): e3102


    Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.

    View details for DOI 10.1371/journal.pone.0003102

    View details for Web of Science ID 000264416600002

    View details for PubMedID 18769617

    View details for PubMedCentralID PMC2518856

  • IAP-IAP complexes required for apoptosis resistance of C. trachomatis-infected cells PLOS PATHOGENS Rajalingam, K., Sharma, M., Paland, N., Hurwitz, R., Thieck, O., Oswald, M., Machuy, N., Rudel, T. 2006; 2 (10): 1013–23


    Host cells infected with obligate intracellular bacteria Chlamydia trachomatis are profoundly resistant to diverse apoptotic stimuli. The molecular mechanisms underlying the block in apoptotic signaling of infected cells is not well understood. Here we investigated the molecular mechanism by which apoptosis induced via the tumor necrosis factor (TNF) receptor is prevented in infected epithelial cells. Infection with C. trachomatis leads to the up-regulation of cellular inhibitor of apoptosis (cIAP)-2, and interfering with cIAP-2 up-regulation sensitized infected cells for TNF-induced apoptosis. Interestingly, besides cIAP-2, cIAP-1 and X-linked IAP, although not differentially regulated by infection, are required to maintain apoptosis resistance in infected cells. We detected that IAPs are constitutively organized in heteromeric complexes and small interfering RNA-mediated silencing of one of these IAPs affects the stability of another IAP. In particular, the stability of cIAP-2 is modulated by the presence of X-linked IAP and their interaction is stabilized in infected cells. Our observations suggest that IAPs are functional and stable as heteromers, a thus far undiscovered mechanism of IAP regulation and its role in modulation of apoptosis.

    View details for DOI 10.1371/journal.ppat.0020114

    View details for Web of Science ID 000242786900013

    View details for PubMedID 17069460

    View details for PubMedCentralID PMC1626104