Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence

The cytoadherence of Trichomonas vaginalis, the sexually transmitted flagellated protozoan, to vaginal epithelial cells (VECs) is the key to infection. Electron microscopy revealed that in vitro-grown parasites having typical globular shape transformed rapidly after contact with VECs into thin, flat, amoeboid cells, maximizing the area of adhesion to the surface of VECs. Amoebic trichomonads formed filopodia and pseudopodia, which interdigitated at distinct sites on the plasma membrane of target cells. In contrast, the amoeboid transformation did not occur for T. vaginalis interacting with He La cells, the previously used in vitro host model cell. Initial parasitism of VECs by a single organism was followed by establishment of a monolayer of trichomonads on the host cell. Finally, parasites adhering to either VECs or HeLa cells were induced to synthesize greater amounts of the four previously described adhesins. Therefore, distinct signals after contact with either epithelial cell type leads to the morphological transformation and/or induction of adhesin synthesis by T. vaginalis.     

Three aspecific ATPases in Trichomonas vaginalis.

1. Three aspecific ATPases were found in the sedimentible fractions of Trichomonas vaginalis. 2. One, with a pH optimum of 5.5, was equally activated by Ca2+ or Mg2+, moderately stable, preferred nucleotide diphosphates as substrates, and was inhibited by vanadate, oligomycin, nitrate and Na+. 3. A second, with a pH optimum of 7.5, was activated by Mg2+, preferred guanosine diphosphate as substrate, and was the least stable and most subject to inhibitors (vanadate, oligomycin, NEM, NBD-Cl, azide and Cl-). 4. The third, pH optimum 8.0, was activated by Ca2+, was latent and the most stable, reacted equally well with nucleotide tri- or diphosphates, and was the least susceptible to inhibitors (vanadate and NEM). 5. All exhibited proton-translocating ability.     

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Prediction and analysis of paralogous proteins in Trichomonas vaginalis genome

Trichomonas vaginalis causes trichomoniasis, second most sexually transmitted disease. The genome sequence draft of T. vaginalis was published by The Institute of Genomic Research reveals an abnormally large genome size of 160 Mb. It was speculated that a significant portion of the proteome contains paralogous proteins. The present study was aimed at identification and analysis of the paralogous proteins. The all against all search approach is used to identify the paralogous proteins. The dataset of proteins was retrieved from TIGR and TrichDB FTP server. The BLAST-P program performed all against all database searches against the protein database of Trichomonas vaginalis available at NCBI genome database. In the present study about 50,000 proteins were searched where 2,700 proteins were found to be paralogous under the rigid selection criteria. The Pfam database search has identified significant number of paralogous proteins which were further categorized among different 1496 paralogous protein in pfam families, 1027 paralogous protein contains domain, 60 proteins were having different repeats and 1092 paralogous protein sequences of clans. Such identification and functional annotation of paralogous proteins will also help in removing paralogous proteins from possible drug targets in future. Presence of huge number of paralogous proteins across wide range of gene families and domains may be one of the possible mechanisms involved in the T. vaginalis genome expansion and evolution.     

A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.     

Trichomonas vaginalis surface proteins: a view from the genome

Surface proteins of mucosal microbial pathogens play multiple and essential roles in initiating and sustaining the colonization of the heavily defended mucosa. The protist Trichomonas vaginalis is one of the most common human sexually transmitted pathogens that colonize the urogenital mucosa. However, little is known about its surface proteins. The recently completed draft genome sequence of T. vaginalis provides an invaluable resource to guide molecular and cellular characterization of surface proteins and to investigate their role in pathogenicity. Here, we review the existing data on T. vaginalis surface proteins and summarize some of the main findings from the recent in silico characterization of its candidate surface proteins.     

The ATP-binding cassette proteins of the deep-branching protozoan parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters.     

阴道毛滴虫症状株和带虫株黏附蛋白33基因的克隆和序列比较

目的分析和比较阴道毛滴虫症状株和带虫株黏附蛋白33基因序列。方法提取阴道毛滴虫各分离株基因组DNA,PCR扩增目的基因,构建重组质粒,克隆,鉴定和序列比较。结果黏附蛋白33基因长度约为930bp,成功构建pMD-18T-ap33重组质粒。同GenBank上的黏附蛋白33基因序列比较,症状株黏附蛋白33基因序列与已知序列有2个碱基不同,而带虫株黏附蛋白33基因序列与已知序列有1个碱基存在差别。结论阴道毛滴虫症状株和带虫株黏附蛋白33基因序列存在差异。     

阴道毛滴虫黏附蛋白33基因原核表达载体的构建及表达

目的构建阴道毛滴虫黏附蛋白33基因原核表达载体并诱导其体外表达。方法pMD-18T-ap33重组质粒和pUC18空质粒经BamH Ⅰ和XbaⅠ限制性内切酶双酶切,将ap33基因亚克隆入pUC18载体并进行筛选和鉴定。重组质粒经IPTG诱导,SDS-PAGE电泳及Western-blot杂交鉴定重组蛋白。结果经双酶切及PCR鉴定,构建的重组质粒为阳性重组子,诱导出的重组蛋白大小约为Mr36000,与理论值基本相符。结论成功构建重组质粒并获得体外表达。     

Visualization of new virus-like-particles in Trichomonas vaginalis

In the present work, we demonstrate virus-like particles (VLPs) with various morphological variations in Trichomonas vaginalis. The VLPs were distinct based on size, shape and electron density, with VLPs being either electron-dense or electron-lucent. We used electron microscopy thin sections of several T. vaginalis strains virus-infected, and also negative staining of fractions obtained after purification by CsCl buoyant density gradient centrifugation. The particles observed in fractions are identical to those previously described, but by thin sections, we found new forms. The shapes found were icosahedral, spherical and oblong, and the sizes varied from 33 to 120nm in diameter with the most common VLP being spherical and having a size range from 83 to 104nm. The VLPs were found in the cytoplasm closely associated with the Golgi complex, with some VLPs budding from the Golgi, and other VLPs were detected adjacent to the plasma membrane. Unidentified cytoplasmic inclusions were observed in the region close to the VLPs and Golgi. Clusters of the already described icosahedral virus were also observed in the cytoplasm, although less frequently. These results indicate that T. vaginalis organisms may be infected with different dsRNA viruses simultaneously.