In Figures 7(a)-7(d) of the published article titled "-Actinin TvACTN3 of Trichomonas vaginalis I... more In Figures 7(a)-7(d) of the published article titled "-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism" [1], we mistakenly used the same strip data in panel (a), lanes 3 and 4; panel (b), lanes 3 and 4; panel (c), lanes 2 and 4; and panel (d), lanes 2 and 3. The corrected new Figure 7 and legend are presented here.
Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, To... more Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatindegrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.
The sequence of a cloned genomic fragment of Trichomonas 6aginalis containing a complete actin ge... more The sequence of a cloned genomic fragment of Trichomonas 6aginalis containing a complete actin gene was determined. An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene. Two overlapped consensus promoter sequences for T. 6aginalis were found 12 nucleotides upstream the actin initiation codon. In addition to actin, two incomplete open reading frames were found at the 5% and 3% ends of the clone. These two sequences are expressed and showed similarity to adenylate cyclase genes and a yeast hypothetical protein. The overall sequence showed a higher G +C content and a lower frequency of repeated sequences in the coding regions when compared with the non-coding regions. A similar unequal nucleotide distribution was found in various T. 6aginalis genes retrieved from data bases.
Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3-UTR of Trichomonas vaginalis TvC... more Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.
Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis requ... more Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis require cysteine proteinase activity. Because of the large number of cysteine proteinases synthesized by T. vaginalis, a ligand assay was used to identify specific proteinases which may selectively target host cells. Two cysteine proteinases from trichomonal extracts with relative molecular masses (Mr) of 65,000 daltons (65-kDa) and 30-kDa were found to avidly bind to HeLa cell and vaginal epithelial cell surfaces. The two proteinases were distinguished by differential inhibition with leupeptin and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). Leupeptin pretreatment of live organisms inhibited the 30-kDa proteinase, which concomitantly reduced or eliminated cytoadherence. The T. vaginalis isolates with low levels of cytoadherence also had diminished or no detectable 30-kDa proteinase activity. On the other hand, TLCK pretreatment inhibited both the 30-kDa and 65-kDa proteinases, which resulted in decreased levels of cytoadherence and totally abolished contact-dependent cytotoxicity. Furthermore, isolates capable of attachment but with little or no cytotoxicity toward HeLa cells had no detectable host cell-bound 65-kDa proteinase. Finally, antiserum generated to each proteinase reacted by indirect immunofluorescence with live organisms, suggesting a surface location for both proteinases. This strategy and use of the ligand assay may permit for the deliniation of the role of two specific T. vaginalis surface proteinases in the properties of cytoadherence and cytotoxicity.
Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patie... more Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patients with trichomoniasis were purified, and VEC parasitism by Trichomonas vaginalis was examined. Trichomonads bound equally well to live or dead VECs, and up to 20% of VECs were parasitized. Trichomonal cytadherence of human VECs was time, temperature, and pH dependent. Saturation binding levels of live trichomonads to VECs gave-2 organisms adherent to parasitized VEC. No differences in cytadherence levels were detected by different isolates to VECs from the same patient compared with adherence to VECs from normal individuals. Trypsinized, live T. vaginalis organisms failed to recognize VECs. A ligand assay identified four adhesin candidates, and only organisms without a prominent immunogen on the surface (negative phenotype) cytadhered to VECs and synthesized the adhesins, confirming the results of a recently published report by us on adherence to HeLa cell monolayers (J. F. Alderete and G. E. Garza, Infect. Immun. 56:28-33, 1988). These data show the ability of T. vaginalis to parasitize human vaginal epithelial cells in a specific receptor-ligand manner.
Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four tri... more Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa {AP23) mediate the interaction of T. vaginaiis with epithelial cells. Fresh isolates, when compared with long-term-grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti-adhesin antibodies reacted by immunobiot only with the respective protein and detected, by indirect jmmunofluorescence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact-dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.
Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagell... more Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65−1 and ap65−2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for both ap65−1 and ap65‐2 genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.
Many bacterial species produce a paracrystalline layer, the surface layer, which completely surro... more Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.
The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like... more 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.
The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like... more 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.
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated ... more Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated ... more Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
In Figures 7(a)-7(d) of the published article titled "-Actinin TvACTN3 of Trichomonas vaginalis I... more In Figures 7(a)-7(d) of the published article titled "-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism" [1], we mistakenly used the same strip data in panel (a), lanes 3 and 4; panel (b), lanes 3 and 4; panel (c), lanes 2 and 4; and panel (d), lanes 2 and 3. The corrected new Figure 7 and legend are presented here.
Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, To... more Toxoplasma gondii can infect all nucleated cells from warm-blooded organisms. After infection, Toxoplasma spreads throughout the body and migrates across biological barriers, such as the intestinal and blood-brain barriers, as well as the placenta in pregnant women. The mechanisms for parasite dissemination are still unknown; however, proteases could play a role as a virulence factor. The aim of this study was to detect and to characterize proteases in whole-cell extracts and in excretion/secretion products from tachyzoites of the RH strain isolated from infected mice. Both fractions were analyzed by gelatin and casein zymography and by azocasein degradation. The biochemical characterization of proteases included standardization of optimal conditions for their activation, such as pH, the presence of cofactors, and a reducing agent. In both fractions, we detected at least nine gelatindegrading metalloproteases in the range of 50 to 290 kDa. The proteases present in the excretion/secretion products were found as soluble proteins and not associated with exosome-like vesicles or other secretory vesicles. Moreover, by using casein zymography, it was possible to detect three serine proteases. Exposure of MDCK cells to excretion/secretion products modified the organization of the cell monolayer, and this effect was reverted after washing thoroughly with PBS and inhibition by metalloprotease and serine protease inhibitors. Proteomic analysis of excretion/secretion products identified 19 proteases. These findings suggest that tachyzoites of a highly virulent strain of Toxoplasma use a battery of proteases to modify the epithelium, probably as a strategy to facilitate their tissue dissemination.
The sequence of a cloned genomic fragment of Trichomonas 6aginalis containing a complete actin ge... more The sequence of a cloned genomic fragment of Trichomonas 6aginalis containing a complete actin gene was determined. An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene. Two overlapped consensus promoter sequences for T. 6aginalis were found 12 nucleotides upstream the actin initiation codon. In addition to actin, two incomplete open reading frames were found at the 5% and 3% ends of the clone. These two sequences are expressed and showed similarity to adenylate cyclase genes and a yeast hypothetical protein. The overall sequence showed a higher G +C content and a lower frequency of repeated sequences in the coding regions when compared with the non-coding regions. A similar unequal nucleotide distribution was found in various T. 6aginalis genes retrieved from data bases.
Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3-UTR of Trichomonas vaginalis TvC... more Iron-Responsive Element of IRE-tvcp12 Hairpin Structure at the 3-UTR of Trichomonas vaginalis TvCP12 mRNA That Binds TvHSP70 and TvACTN-3 Can Regulate mRNA Stability and Amount of Protein.
Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis requ... more Recent reports strongly suggest that cytoadherence and cytotoxicity by Trichomonas vaginalis require cysteine proteinase activity. Because of the large number of cysteine proteinases synthesized by T. vaginalis, a ligand assay was used to identify specific proteinases which may selectively target host cells. Two cysteine proteinases from trichomonal extracts with relative molecular masses (Mr) of 65,000 daltons (65-kDa) and 30-kDa were found to avidly bind to HeLa cell and vaginal epithelial cell surfaces. The two proteinases were distinguished by differential inhibition with leupeptin and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). Leupeptin pretreatment of live organisms inhibited the 30-kDa proteinase, which concomitantly reduced or eliminated cytoadherence. The T. vaginalis isolates with low levels of cytoadherence also had diminished or no detectable 30-kDa proteinase activity. On the other hand, TLCK pretreatment inhibited both the 30-kDa and 65-kDa proteinases, which resulted in decreased levels of cytoadherence and totally abolished contact-dependent cytotoxicity. Furthermore, isolates capable of attachment but with little or no cytotoxicity toward HeLa cells had no detectable host cell-bound 65-kDa proteinase. Finally, antiserum generated to each proteinase reacted by indirect immunofluorescence with live organisms, suggesting a surface location for both proteinases. This strategy and use of the ligand assay may permit for the deliniation of the role of two specific T. vaginalis surface proteinases in the properties of cytoadherence and cytotoxicity.
Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patie... more Human vaginal epithelial cells (VECs) from vaginal swabs obtained from normal women or from patients with trichomoniasis were purified, and VEC parasitism by Trichomonas vaginalis was examined. Trichomonads bound equally well to live or dead VECs, and up to 20% of VECs were parasitized. Trichomonal cytadherence of human VECs was time, temperature, and pH dependent. Saturation binding levels of live trichomonads to VECs gave-2 organisms adherent to parasitized VEC. No differences in cytadherence levels were detected by different isolates to VECs from the same patient compared with adherence to VECs from normal individuals. Trypsinized, live T. vaginalis organisms failed to recognize VECs. A ligand assay identified four adhesin candidates, and only organisms without a prominent immunogen on the surface (negative phenotype) cytadhered to VECs and synthesized the adhesins, confirming the results of a recently published report by us on adherence to HeLa cell monolayers (J. F. Alderete and G. E. Garza, Infect. Immun. 56:28-33, 1988). These data show the ability of T. vaginalis to parasitize human vaginal epithelial cells in a specific receptor-ligand manner.
Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four tri... more Parasitism of host epithelial cells by Trichomonas vaginalis is a highly specific event. Four trichomonad surface proteins (adhesins) with molecular masses of 65000 daltons (65kDa; AP65), 51 kDa (AP51), 33kDa (AP33), and 23 kDa {AP23) mediate the interaction of T. vaginaiis with epithelial cells. Fresh isolates, when compared with long-term-grown isolates, had greater amounts of adhesins, which corresponded with increased levels of cytoadherence. Anti-adhesin antibodies reacted by immunobiot only with the respective protein and detected, by indirect jmmunofluorescence, each adhesin on the parasite surface. These antibodies inhibited the binding of live parasites to epithelial cells and protected epithelial cells from contact-dependent cytotoxicity. The pretreatment of epithelial cells with a preparation of purified adhesins also blocked trichomonal cytoadherence. Moreover, HeLa cells possessed molecules which recognized and bound to adhesins on nitrocellulose blots.
Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagell... more Cytoadherence to the vaginal epithelium is a critical step in infection by the eukaryotic flagellate Trichomonas vaginalis. Four trichomonad surface proteins (AP65, AP51, AP33 and AP23) mediate cytoadherence. The cDNA encoding the AP65 adhesin was isolated from a phagemid cDNA expression library by screening with antiserum and monoclonal antibody (mAb) raised against the purified trichomonad AP65 protein. Two clones, F11.2 and F11.5, coded for immuno‐crossreactive recombinant proteins that possessed functional properties equal to the T. vaginalis AP65 adhesin. Analysis of full‐length sequences corresponding to the F11.2 and F11.5 cDNAs revealed that both contained 1701‐base open reading frames (ORFs) that encoded proteins of 63 281 daltons and 63087 daltons, respectively. Comparison of the full‐length sequences showed 87% identity at the nucleotide level and 91% identity at the protein level. Restriction‐enzyme mapping and Southern analysis reaffirmed the distinctness of the F11.2 and F11.5 cDNAs, indicating that two different AP65 genes (now called ap65−1 and ap65−2) are present in the T. vaginalis genome in at least two copies each. Northern analysis detected high levels of transcript of ∼1.8 kb for both ap65−1 and ap65‐2 genes in trichomonads grown only in high‐iron medium, confirming the transcriptional regulation of adhesin synthesis by iron. Homology searches revealed significant similarity (38% amino acid identity and 54% nucleotide identity) to malic enzymes. However, purified malic enzyme and mAb to AP65 crossreactive with malic enzyme neither inhibited cytoadherence of T. vaginalis to host cells nor prevented binding of the trichomonad AP65 to HeLa cells in a ligand assay.
Many bacterial species produce a paracrystalline layer, the surface layer, which completely surro... more Many bacterial species produce a paracrystalline layer, the surface layer, which completely surrounds the exterior of the cell. In some bacteria, the surface layer is implicated in pathogenesis. Two proteins present in cell wall extracts from Clostridium tetani have been investigated and identified one of these has been unambiguously as the surface-layer protein (SLP). The gene, slpA, has been located in the genome of C. tetani E88 that encodes the SLP. The molecular mass of the protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is considerably larger than that predicted from the gene; however the protein does not appear to be glycosylated. Furthermore, analysis of five C. tetani strains, including three recent clinical isolates, shows considerable variation in the sizes of the SLP.
The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like... more 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.
The recombinant TvCP4 prepro region (ppTvCP4r) acts as an exogenous inhibitor of cathepsin L-like... more 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.
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated ... more Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated ... more Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.
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