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Rossana Arroyo - One of the best experts on this subject based on the ideXlab platform.
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characterization of a novel endogenous cysteine proteinase inhibitor trichocystatin 3 tc 3 localized on the surface of Trichomonas vaginalis
The International Journal of Biochemistry & Cell Biology, 2018Co-Authors: Diana Belen Sanchezrodriguez, Jaime Ortegalopez, Rosa Elena Cardenasguerra, Gerardo Resendizcardiel, Bibiana Chavezmunguia, Anel Lagunesguillen, Rossana ArroyoAbstract:Abstract Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.
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Dataset of cathepsin L-like CP inhibition of Naegleria fowleri and Acanthamoeba castellanii by ppTvCP4r from Trichomonas vaginalis
Elsevier, 2018Co-Authors: Rosa E. Cárdenas-guerra, Moisés Martínez-castillo, Jaime Ortega-lópez, Mineko Shibayama, Rossana ArroyoAbstract:The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like CPs from Trichomonas vaginalis (Cárdenas-Guerra et al., 2015 [1]). Here, we present the dataset of the trichomonad ppTvCP4r inhibitory effect against the CP proteolytic activities from other microorganisms, such as Naegleria fowleri and Acanthamoeba castellanii free-living amoeba. The proteolytic activity inhibition of total crude extracts (TCEs) of N. fowleri and A. castellanii was determined and recorded using a fluorogenic substrate specific for cathepsin L CPs without or with a ppTvCP4r treatment at different concentrations and pH. Keywords: Acanthamoeba castellanii, Cathepsin L-like CPs, Cysteine proteinase inhibitors, Naegleria fowleri, ppTvCP4r, Trichomonas vaginali
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the tvlegu 1 a legumain like cysteine proteinase plays a key role in Trichomonas vaginalis cytoadherence
BioMed Research International, 2013Co-Authors: Francisco Javier Rendongandarilla, Marlene Benchimol, Jaime Ortegalopez, Lucero A Ramonluing, Ivone Rosa De Andrade, Rossana ArroyoAbstract:The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.
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immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis
Proteomics, 2010Co-Authors: Lucero A Ramonluing, Leticia Avilagonzalez, Jaime Ortegalopez, Francisco Javier Rendongandarilla, Rosa Elena Cardenasguerra, Norma A Rodriguezcabrera, Claudia Angelortiz, Carmen N Herrerasanchez, Manuela Mendozagarcia, Rossana ArroyoAbstract:Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.
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the trichomonad cysteine proteinase tvcp4 transcript contains an iron responsive element
FEBS Letters, 2007Co-Authors: Eduardo Solanogonzalez, Leticia Avilagonzalez, Jaime Ortegalopez, Eduviges Burrolabarraza, Claudia R Leonsicairos, Lorena Gutierrezescolano, Rossana ArroyoAbstract:The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron-responsive element (IRE)-like stem-loop structure at the 5'-region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post-transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP-like system. Gel-shifting, UV cross-linking and competition experiments demonstrated that this IRE-like structure specifically bound to human iron regulatory protein-1. IRP-like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low-iron conditions were also found in trichomonads. Thus, a post-transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP-like interactions was found.
Eric Viscogliosi - One of the best experts on this subject based on the ideXlab platform.
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prevalence risk factors of infection and molecular characterization of trichomonads in puppies from french breeding kennels
Veterinary Parasitology, 2013Co-Authors: Aurélien Grellet, Laurine Vandewynckel, Amandine Cian, Dionigia Meloni, Corine Boucrautbaralon, Didier Grandjean, Alexandre Feugier, Eric ViscogliosiAbstract:Abstract The trichomonad species TriTrichomonas fetus and PentaTrichomonas hominis were recently identified in the feces of dogs with diarrhea. However the prevalence and pathogenicity of these parasites in the canine population still remained poorly resolved. Therefore the aim of the present study was (1) to determine the prevalence of trichomonads infecting puppies living in French breeding kennels, (2) to confirm the predominance of P. hominis in dogs, (3) to investigate the genetic diversity of P. hominis isolates identified in the French canine population and (4) to evaluate the risk factors for infection by P. hominis and the influence of the parasite on feces consistency. A total of 215 both diarrheic and non-diarrheic puppies from 25 French breeding kennels were included in this epidemiological survey. Fecal samples from each puppy were examined for 6 gastrointestinal pathogens: parvovirus type 2 (CPV2), coronavirus, Toxocara canis, Cystoisospora ohioensis-complex, Cystoisospora canis, and Giardia intestinalis. A part of each collected stool was also tested for the presence of motile trichomonads by microscopy after culturing. The prevalence of trichomonad infection was 15.8% (34/215) among puppies and 20% (5/25) among breeding kennels. DNA from 26 of the 34 positive samples was successfully amplified using a trichomonad-specific primer pair. Analysis of the sequences of PCR products indicated that P. hominis was the only trichomonad infecting the canine population. All the puppies infected with P. hominis belonged to large breed dogs. Moreover, puppies from large breeding kennels, excreting a high level of G. intestinalis and/or excreting a high level of C. canis oocysts showed a higher probability of being positive for P. hominis infection. Univariate analysis also revealed an increased risk for P. hominis infection in puppies with abnormal feces. However, in a multivariate analysis, CPV2 was the only gastrointestinal pathogen associated with abnormal feces. Since enteropathogens were commonly found in dogs infected by P. hominis, the pathogenic potential of this trichomonad species remained uncertain and has to be further evaluated by experimental infection studies.
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phylogenetic relationships of class ii fumarase genes from trichomonad species
Molecular Biology and Evolution, 2001Co-Authors: Delphine Gerbod, Jan Tachezy, Christophe Noel, Virginia P Edgcomb, Stepanka Vanacova, Rene Wintjens, Mitchell L Sogin, Eric ViscogliosiAbstract:Class II fumarase sequences were obtained by polymerase chain reaction from five trichomonad species. All residues known to be highly conserved in this enzyme were present. Nuclear run-on assays showed that one of the two genes identified in TriTrichomonas foetus was expressed, whereas no fumarase transcripts were detected in the related species Trichomonas vaginalis. These findings corroborate previous biochemical data. Fumarase genes were also expressed in Monocercomonas sp. and TetraTrichomonas gallinarum but not in PentaTrichomonas hominis, Trichomonas gallinae, Trichomonas tenax, and Trichomitus batrachorum under the culture conditions used. Molecular trees inferred by likelihood methods reveal that trichomonad sequences have no affinity to described class II fumarase genes from other eukaryotes. The absence of functional mitochondria in protists such as trichomonads suggests that they diverged from other eukaryotes prior to the alpha-proteobacterial symbiosis that led to mitochondria. Furthermore, they are basal to other eukaryotes in rRNA analyses. However, support for the early-branching status of trichomonads and other amitochondriate protists based on phylogenetic analyses of multiple data sets has been equivocal. Although the presence of hydrogenosomes suggests that trichomonads once had mitochondria, their class II iron-independent fumarase sequences differ markedly from those of other mitochondriate eukaryotes. All of the class II fumarase genes described from other eukaryotes are of apparent alpha-proteobacterial origin and hence a marker of mitochondrial evolution. In contrast, the class II fumarase from trichomonads emerges among other eubacterial homologs. This is intriguing evidence for an independent acquisition of these genes in trichomonads apart from the mitochondrial endosymbiosis event that gave rise to the form present in other eukaryotes. The ancestral trichomonad class II fumarase may represent a prokaryotic form that was replaced in other eukaryotes after the divergence of trichomonads with the movement of endosymbiont genes into the nucleus. Alternatively, it may have been acquired via a separate endosymbiotic event or lateral gene transfer.
Jaime Ortegalopez - One of the best experts on this subject based on the ideXlab platform.
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characterization of a novel endogenous cysteine proteinase inhibitor trichocystatin 3 tc 3 localized on the surface of Trichomonas vaginalis
The International Journal of Biochemistry & Cell Biology, 2018Co-Authors: Diana Belen Sanchezrodriguez, Jaime Ortegalopez, Rosa Elena Cardenasguerra, Gerardo Resendizcardiel, Bibiana Chavezmunguia, Anel Lagunesguillen, Rossana ArroyoAbstract:Abstract Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.
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the tvlegu 1 a legumain like cysteine proteinase plays a key role in Trichomonas vaginalis cytoadherence
BioMed Research International, 2013Co-Authors: Francisco Javier Rendongandarilla, Marlene Benchimol, Jaime Ortegalopez, Lucero A Ramonluing, Ivone Rosa De Andrade, Rossana ArroyoAbstract:The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.
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immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis
Proteomics, 2010Co-Authors: Lucero A Ramonluing, Leticia Avilagonzalez, Jaime Ortegalopez, Francisco Javier Rendongandarilla, Rosa Elena Cardenasguerra, Norma A Rodriguezcabrera, Claudia Angelortiz, Carmen N Herrerasanchez, Manuela Mendozagarcia, Rossana ArroyoAbstract:Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.
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the trichomonad cysteine proteinase tvcp4 transcript contains an iron responsive element
FEBS Letters, 2007Co-Authors: Eduardo Solanogonzalez, Leticia Avilagonzalez, Jaime Ortegalopez, Eduviges Burrolabarraza, Claudia R Leonsicairos, Lorena Gutierrezescolano, Rossana ArroyoAbstract:The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron-responsive element (IRE)-like stem-loop structure at the 5'-region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post-transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP-like system. Gel-shifting, UV cross-linking and competition experiments demonstrated that this IRE-like structure specifically bound to human iron regulatory protein-1. IRP-like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low-iron conditions were also found in trichomonads. Thus, a post-transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP-like interactions was found.
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a Trichomonas vaginalis 120 kda protein with identity to hydrogenosome pyruvate ferredoxin oxidoreductase is a surface adhesin induced by iron
Cellular Microbiology, 2004Co-Authors: Veronica Morenobrito, Leticia Avilagonzalez, Jaime Ortegalopez, Carmina Yanezgomez, Patricia Mezacervantez, Mario A Rodriguez, Arturo Gonzalezrobles, Rossana ArroyoAbstract:Summary Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 µM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.
Marlene Benchimol - One of the best experts on this subject based on the ideXlab platform.
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the tvlegu 1 a legumain like cysteine proteinase plays a key role in Trichomonas vaginalis cytoadherence
BioMed Research International, 2013Co-Authors: Francisco Javier Rendongandarilla, Marlene Benchimol, Jaime Ortegalopez, Lucero A Ramonluing, Ivone Rosa De Andrade, Rossana ArroyoAbstract:The goal of this paper was to characterize a Trichomonas vaginalis cysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor during T. vaginalis infection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.
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Trichomonas vaginalis and triTrichomonas foetus interaction with fibroblasts and muscle cells new insights into parasite mediated host cell cytotoxicity
Memorias Do Instituto Oswaldo Cruz, 2012Co-Authors: Ricardo Chaves Vilela, Marlene BenchimolAbstract:Trichomonas vaginalis and TriTrichomonas foetus are parasitic, flagellated protists that inhabit the urogenital tract of humans and bovines, respectively. T. vaginalis causes the most prevalent non-viral sexually transmitted disease worldwide and has been associated with an increased risk for human immunodeficiency virus-1 infection in humans. Infections by T. foetus cause significant losses to the beef industry worldwide due to infertility and spontaneous abortion in cows. Several studies have shown a close association between trichomonads and the epithelium of the urogenital tract. However, little is known concerning the interaction of trichomonads with cells from deeper tissues, such as fibroblasts and muscle cells. Published parasite-host cell interaction studies have reported contradictory results regarding the ability of T. foetus and T. vaginalis to interact with and damage cells of different tissues. In this study, parasite-host cell interactions were examined by culturing primary human fibroblasts obtained from abdominal biopsies performed during plastic surgeries with trichomonads. In addition, mouse 3T3 fibroblasts, primary chick embryo myogenic cells and L6 muscle cells were also used as models of target cells. The parasite-host cell cultures were processed for scanning and transmission electron microscopy and were tested for cell viability and cell death. JC-1 staining, which measures mitochondrial membrane potential, was used to determine whether the parasites induced target cell damage. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling staining was used as an indicator of chromatin damage. The colorimetric crystal violet assay was performed to ana-lyse the cytotoxicity induced by the parasite. The results showed that T. foetus and T. vaginalis adhered to and were cytotoxic to both fibroblasts and muscle cells, indicating that Trichomonas infection of the connective and muscle tissues is likely to occur; such infections could cause serious risks to the infected host.
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Trichomonas vaginalis kills and eats evidence for phagocytic activity as a cytopathic effect
Parasitology, 2010Co-Authors: Victor Midlej, Marlene BenchimolAbstract:This study reports that the cytopathic effect of Trichomonas vaginalis, an important human parasite of the urogenital tract, occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions.
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The hydrogenosome as a drug target.
Current Pharmaceutical Design, 2008Co-Authors: Marlene BenchimolAbstract:: Hydrogenosomes are spherical or slightly elongated organelles found in non-mitochondrial organisms. In Trichomonas hydrogenosomes measure between 200 to 500 nm, but under drug treatment they can reach 2 microm. Like mitochondria hydrogenosomes: (1) are surrounded by two closely apposed membranes and present a granular matrix: (2) divide in three different ways: segmentation, partition and the heart form; (3) they may divide at any phase of the cell cycle; (4) produce ATP; (5) participate in the metabolism of pyruvate formed during glycolysis; (6) are the site of molecular hydrogen formation; (7) present a relationship with the endoplasmic reticulum; (8) incorporate calcium; (9) import proteins post-translationally; (10) present cardiolipin. However, there are differences, such as: (1) absence of genetic material, at least in Trichomonas; (2) lack a respiratory chain and cytochromes; (3) absence of the F(0)-F(1) ATPase; (4) absence of the tricarboxylic acid cycle; (5) lack of oxidative phosphorylation; (6) presence of peripheral vesicles. Hydrogenosomes are considered an excellent drug target since their metabolic pathway is distinct from those found in mitochondria and thus medicines directed to these organelles will probably not affect the host-cell. The main drug used against trichomonads is metronidazole, although other drugs such as beta-Lapachone, colchicine, Taxol, nocodazole, griseofulvin, cytochalasins, hydroxyurea, among others, have been used in trichomonad studies, showing: (1) flagella internalization forming pseudocyst; (2) dysfunctional hydrogenosomes; (3) hydrogenosomes with abnormal sizes and shapes and with an electron dense deposit called nucleoid; (4) intense autophagy in which hydrogenosomes are removed and further digested in lysosomes.
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Trichomonas vaginalis harboring Mycoplasma hominis increases cytopathogenicity in vitro
European Journal of Clinical Microbiology & Infectious Diseases, 2007Co-Authors: R. G. Vancini, A. Pereira-neves, R. Borojevic, Marlene BenchimolAbstract:The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. Mycoplasmas are frequently found with trichomonads but the consequences of this association are not yet known. In the present study, the effects of T. vaginalis harboring M. hominis on human vaginal epithelial cells and on MDCK cells are described. The results were analyzed by light, scanning and transmission electron microscopy, as well as using cell viability assays. There was an increase in the cytopathic effects on the epithelial cells infected with T. vaginalis associated with M. hominis compared to T. vaginalis alone. The epithelial cells exhibited an increase in the intercellular spaces, a lesser viability, and increased destruction provoked by the infected T. vaginalis . In addition, the trichomonads presented a higher amoeboid transformation rate and an intense phagocytic activity, characteristics of higher virulence behavior.
Leticia Avilagonzalez - One of the best experts on this subject based on the ideXlab platform.
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immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis
Proteomics, 2010Co-Authors: Lucero A Ramonluing, Leticia Avilagonzalez, Jaime Ortegalopez, Francisco Javier Rendongandarilla, Rosa Elena Cardenasguerra, Norma A Rodriguezcabrera, Claudia Angelortiz, Carmen N Herrerasanchez, Manuela Mendozagarcia, Rossana ArroyoAbstract:Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D-zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200-21 kDa of parasite protease-resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis-positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU-1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis.
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the trichomonad cysteine proteinase tvcp4 transcript contains an iron responsive element
FEBS Letters, 2007Co-Authors: Eduardo Solanogonzalez, Leticia Avilagonzalez, Jaime Ortegalopez, Eduviges Burrolabarraza, Claudia R Leonsicairos, Lorena Gutierrezescolano, Rossana ArroyoAbstract:The differential expression of the Trichomonas vaginalis cysteine proteinase TVCP4 by iron at the protein synthesis level and the prediction of an iron-responsive element (IRE)-like stem-loop structure at the 5'-region of the T. vaginalis cysteine proteinase 4 gene (tvcp4) mRNA suggest a post-transcriptional mechanism of iron regulation in trichomonads mediated by an IRE/IRP-like system. Gel-shifting, UV cross-linking and competition experiments demonstrated that this IRE-like structure specifically bound to human iron regulatory protein-1. IRP-like cytoplasmic proteins that bound human ferritin IRE sequence transcripts at low-iron conditions were also found in trichomonads. Thus, a post-transcriptional regulatory mechanism by iron for tvcp4 mediated by IRE/IRP-like interactions was found.
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a Trichomonas vaginalis 120 kda protein with identity to hydrogenosome pyruvate ferredoxin oxidoreductase is a surface adhesin induced by iron
Cellular Microbiology, 2004Co-Authors: Veronica Morenobrito, Leticia Avilagonzalez, Jaime Ortegalopez, Carmina Yanezgomez, Patricia Mezacervantez, Mario A Rodriguez, Arturo Gonzalezrobles, Rossana ArroyoAbstract:Summary Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 µM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.
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cp30 a cysteine proteinase involved in Trichomonas vaginalis cytoadherence
Infection and Immunity, 2000Co-Authors: M R Mendozalopez, Leticia Avilagonzalez, Cecilia Becerrilgarcia, Loriz Viridiana Fattelfacenda, Jaime Ortegalopez, M E Ruiztachiquin, Rossana ArroyoAbstract:We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.
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a novel cysteine proteinase cp65 of Trichomonas vaginalis involved in cytotoxicity
Microbial Pathogenesis, 2000Co-Authors: Maria Elizbeth Alvarezsanchez, Leticia Avilagonzalez, Cecilia Becerrilgarcia, Loriz Viridiana Fattelfacenda, Jaime Ortegalopez, Rossana ArroyoAbstract:Abstract The goal of this study was to demonstrate the participation in cellular damage of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa (CP65). By two dimensional gelatin-gel electrophoresis of trichomonad proteins we detected four spots with proteolytic activity on the 65 kDa region, but only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluorescence, rabbit antibodies against this proteinase localized the CP65 on the plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of parasites with the specific anti-CP65 antibody reduced trichomonal cytotoxicity to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3-carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) abrogated the proteinase activity and reduced cytotoxicity levels of T. vaginalis in cell culture monolayers, indicating that the trichomonad CP65 is a cysteine proteinase. Activity of the CP65 proteinase was optimal at pH 5.5 and 37°C, conditions similar to those of patients with trichomonosis. Also, this proteinase degraded some of the proteins found in the vagina, i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Finally, immunoprecipitation assays showed that sera and vaginal washes from trichomonosis patient possess anti-CP65 antibodies. In conclusion, results presented in this work demonstrate that the CP65 is a surface cysteine proteinase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a virulence factor. It is immunogenic during human infection and degrades some extracellular matrix proteins, i.e. collagen IV and fibronectin.
