Dataset Information

Trichomonas vaginalis

ABSTRACT: Trichomonas vaginalis is a sexually transmitted infection that causes vaginitis and increases the risk of HIV transmission. We are interested in the secreted and membrane glycoproteins of Trichomonas because they are likely involved in pathogenesis and may include novel vaccine targets. Four mass spectrometric methods (identification of all parasite proteins, glycoprotein enrichment with the plant lectin Concanavalin A, peptide:N-glycanase treatment to identify occupied N-glycans sites, and analysis of N-terminal peptides) were used to identify >300 Trichomonas secreted and membrane proteins. The first group of these proteins, which were present in multiple genome copies and had homologs in diverse eukaryotes, included 1) those involved in the N-glycan-dependent quality control protein folding in the ER lumen, 2) metalloproteases, serine proteases, cysteine proteases, and other lysosomal enzymes, and 3) transporters and membrane-associated cyclases. The second group of secreted and membrane proteins were, for the most part, encoded by single copy genes, unique to Trichomonas, and missing N-terminal signal peptides. The latter observation is despite evidence that the signal peptide peptidase functions normally in Trichomonas. As the unique secreted and membrane proteins of Trichomonas were often large and lacked features that make it easy to choose vaccine candidates, alternative strategies for vaccination and/or therapy are discussed.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Trichomonas Vaginalis Eukaryota (eucaryotes)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Trichomoniasis

SUBMITTER: Andrea Carpentieri

LAB HEAD: John Crawford Samuelson

PROVIDER: PXD000699 | Pride | 2022-02-22

REPOSITORIES: Pride

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Publications

Gene expansion in Trichomonas vaginalis: a case study on transmembrane cyclases.

Cui Jike J Smith Temple F TF Samuelson John J

Genome informatics. International Conference on Genome Informatics 20070101

The draft genome of Trichomonas vaginalis was recently published, but not much is known on why it has such a large genome. In part this size is due to many gene family expansions. For example we found over 100 members in the adenylyl cyclase family. About half are complete full length genes, and nearly half are initially confirmed to be pseudogenes, the remaining are either incomplete or the apparent result of assembly or sequencing problems. The family can be divided into two subgroups by seque ...[more]

PMID: 18546472

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Project description:Lysosome represent a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several lysosomal proteins, including the cysteine protease TvCP2, were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what the precise structure of the lysosomal recognition marker needs to be clarified in future studies.

Project description:Mitochondria originated from α-proteobacterial endosymbionts, and the endosymbiont-to-organelle transition is tightly linked to the establishment of protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, recent studies have shown diversity of TOM subunits between main eukaryotic lineages. However, experimental knowledge is currently limited to a few model organisms. Here, we analysed the TvTOM complex in hydrogenosomes, an anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. Using the pore-forming TvTom40-2 as bait, we immunoprecipitated the high molecular weight TvTOM complex both under crosslinking and native conditions and performed label-free quantitative mass spectrometry (LFQ-MS) to identify the components of the TvTOM complex. Identification of the components of the hydrogenosomal TOM complex To identify interaction partners for TvTom40-2 in the TOM complex, we performed co-immunoprecipitations (coIPs) of HA-tagged TvTom40-2 under protein crosslinking and under native conditions, and the eluted proteins were analyzed using label-free quantitative (LFQ) mass spectrometry. CoIPs using anti-HA antibody (Ab) were performed with hydrogenosomes isolated from both the strain expressing HA-tagged TvTom40-2 and the wild type strain, as a negative control. The analysis revealed 50 and 36 proteins that were enriched with HA-tagged TvTom40-2 under crosslinking and native conditions respectively. As TOM proteins are embedded in the hydrogenosomal outer membrane, we searched for proteins with TMDs in the datasets using TMHMM and found 19 and 13 proteins for crosslinking and native coIPs respectively. The intersection between the two datasets and the hydrogenosomal membrane proteome (Rada et al 2011) contained five TvTom40 isoforms, two TA proteins named Homp36 and Homp19 (Homp, Hydrogenosomal outer membrane protein), Sam50 and its paralogue Sam50p, and Hmp35. In addition, the intersection between the coIP dataset under crosslinking conditions and the membrane proteome contained two more TA proteins, Homp38 and Homp46. Based on these results, we selected Homp36 which was previously shown to reside in the hydrogenosomal outer membrane (Rada et al 2011) for the reciprocal coIPs. Proteins enriched in the HA-tagged Homp36 coIPs under crosslinking conditions included multiple isoforms of TvTom40, Sam50, Hmp35, Homp38, Homp46 and Homp19, and multiple isoforms of TvTom40, Sam50 and Hmp35 were enriched under native conditions. Altogether, the coIP and MS data indicated four TA candidate proteins, Homp19, Homp36, Homp38 and Homp46, and two β-barrel proteins, Sam50 and Hmp35. InterProScan predicted that Homp36, Homp38 and Homp46 carry an N-terminal Hsp20-like chaperone domain, three TPR-like domains and a C-terminal transmembrane helix. This domain architecture resembles the recently reported ATOM69 in T. brucei (Mani et al 2015). Indeed, HHpred searches using Homp36 and Homp38 as queries against the T. brucei proteome revealed ATOM69 as the first hit, with E values of 4.9e-17 and 2.3e-11 respectively. HHpred searches with Homp46 recognized various proteins with TPR domains, whereas no significant homology was observed for Homp19.

Project description:Lysosomes represent a central degradative compartment of eukaryotes, yet little is known about biogenesis and function of this organelle in parasitic protists. Whereas the mannose-6 phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide independent sorting was reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, protein targeting and involvement of lysosomes in hydrolase secretion. The organelles were purified using conventional Percoll and OptiPrep density gradient centrifugation, and a novel purification protocol based on phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in the lysosomal proteome of 462 proteins, which were sorted into 21 functional classes. Hydrolases represent the largest functional class and include proteases, lipases, phosphatases, glycosidases, and other hydrolases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate high dynamics of phagolysosomal compartment. Several lysosomal proteins including the cysteine protease TvCP2 were previously shown to be secreted. Our experiment showed that secretion of TvCP2 was strongly inhibited by chloroquine that increased intralysosomal pH and thus indicated TvCP2 secretion through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified in the phagolysosomal proteome divergent homologs of M6P receptor TvMPR, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP that possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as recognition marker for lysosomal hydrolases. Whether TvMPR or other possible receptors are involved in this process and what is the precise structure of the lysosomal recognition marker need to be clarified in future studies.

Dataset Information

Trichomonas vaginalis

Publications

Gene expansion in Trichomonas vaginalis: a case study on transmembrane cyclases.

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