Chromosomes of parasitic protist Trichomonas vaginalis

Charles University in Prague Faculty of Science Programme of study: Parasitology Abstract of Ph.D. thesis Chromosomes of parasitic protist Trichomonas vaginalis Zuzana Zubáčová, MSc. Supervisor: Prof. RNDr. Jan Tachezy, Ph.D. Praha, 2011 Abstract Genome sequencing and associated proteome projects has revolutionized research in the field of molecular parasitology. Progress in sequencing of parasite as well as free-living species enables comparative and phylogenetic studies and provides important data sets for understanding of basic biology and identification of new drug targets. The genome sequencing of Trichomonas vaginalis revealed surprisingly large genome size of this organism (~160 Mb) that resulted from expansion of various repetitive elements, specific gene families and large scale genome duplication. I studied genome sizes of other nine selected species from Trichomonadea group to find whether other trichomonads have undergone similar genome expansion. The measurement of nuclear DNA content by flow cytometry revealed relatively large DNA content in all tested species although within a broad range (86-177 Mb). The largest genomes were observed in the Trichomonas and Tritrichomonas genera while Tetratrichomonas contains the smallest genome. The genome sizes correlated with the cell volume however no..

Studium chromosomů parazita Trichomonas vaginalis

Charles University in Prague Faculty of Science Programme of study: Parasitology Abstract of Ph.D. thesis Chromosomes of parasitic protist Trichomonas vaginalis Zuzana Zubáčová, MSc. Supervisor: Prof. RNDr. Jan Tachezy, Ph.D. Praha, 2011 Abstract Genome sequencing and associated proteome projects has revolutionized research in the field of molecular parasitology. Progress in sequencing of parasite as well as free-living species enables comparative and phylogenetic studies and provides important data sets for understanding of basic biology and identification of new drug targets. The genome sequencing of Trichomonas vaginalis revealed surprisingly large genome size of this organism (~160 Mb) that resulted from expansion of various repetitive elements, specific gene families and large scale genome duplication. I studied genome sizes of other nine selected species from Trichomonadea group to find whether other trichomonads have undergone similar genome expansion. The measurement of nuclear DNA content by flow cytometry revealed relatively large DNA content in all tested species although within a broad range (86-177 Mb). The largest genomes were observed in the Trichomonas and Tritrichomonas genera while Tetratrichomonas contains the smallest genome. The genome sizes correlated with the cell volume however no...Univerzita Karlova v Praze Přírodovědecká fakulta Program: Biomedicína/Parazitologie Abstrakt disertační práce Chromosomy parazita Trichomonas vaginalis Mgr. Zuzana Zubáčová Školitel: Prof. RNDr. Jan Tachezy, Ph.D. Abstrakt Sekvenování genomů a přidružené proteomické studie způsobily převrat ve výzkumu v oblasti molekulární parazitologie. Postupující sekvenování jak parazitických, tak i volně žijících druhů umožňuje srovnávací a fylogenetické studie, a je rovněž zdrojem důležitých dat pro pochopení jejich základní biologie a identifikaci nových cílů terapie parazitárních nákaz. Sekvenování DNA parazita Trichomonas vaginalis překvapivě odhalilo u tohoto lidského patogena genom o velikosti až ~160 Mb. Zjistilo se, že expanze genomu je výsledkem zmnožení repetitivních sekvencí, specifických genových rodin a duplikací rozsáhlých částí genomu. Analýzou obsahu jaderné DNA u 9 příbuzných druhů trichomonád pomocí průtokové cytometrie jsem zjistila, že expanze velikosti genomu není specifická jen pro T. vaginalis, ale i ostatní druhy se vyznačují velkými genomy (86-177 Mb). Největší genomy mají zástupci rodů Trichomonas a Tritrichomonas, nejmenší genom jsme zjistili u druhu Tetratrichomonas gallinarum. Velikosti genomů trichomonád pozitivně korelovaly s objemy jejich buněk. Nepozorovali jsme korelaci mezi velikosti...Department of ParasitologyKatedra parazitologieFaculty of SciencePřírodovědecká fakult

The Mitochondrion-Like Organelle of <em>Trimastix pyriformis</em> Contains the Complete Glycine Cleavage System

<div><p>All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in <i>Trimastix pyriformis,</i> a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins – mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of <i>Trimastix</i> but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of <i>Trimastix</i>. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of <i>Trimastix pyriformis</i>.</p> </div

Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes

Background: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. Results: We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. Conclusions: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.Science, Faculty ofOther UBCNon UBCBotany, Department ofReviewedFacult

H-protein of GCS localizes into vesicles (putative mitochondrion-like organelles) in <i>Trimastix pyriformis</i>.

<p>A) Immunofluorescence microscopy of the <i>Trimastix pyriformis</i> cell. The green signal from antiH-protein (human) co-localizes with red signal from the antiH-protein (<i>Trimastix</i>). The DNA is stained blue with Hoechst. B) Western blot on the cellular fractions of <i>Trimastix pyriformis.</i> The lines represent pure bacteria <i>Citrobacter</i> sp. from the culture (Bact), high speed pellet of <i>Trimastix</i> (HSP), supernatant of <i>Trimastix</i> (Sup), total lysate of <i>Trimastix</i> (Total).</p

Over-expression of <i>Trimastix</i> proteins in yeast.

<p>The over-expression of GFP tagged proteins of <i>Trimastix</i> in <i>Saccharomyces cerevisiae</i>. The columns represent the signals from GFP tag (green), MitoTracker (red), merged GFP and MitoTracker and DIC. Rows represent individual proteins: cpn60, P1-protein of GCS, H-protein of GCS and H-protein of GCS truncated of the first 16 amino acids.</p