Proteomic profile approach of effect of putrescine depletion over Trichomonas vaginalis.

Infection with Trichomonas vaginalis produces a malodorous seropurulent vaginal discharge due to several chemicals, including polyamines. The presence of 1,4-diamino-2-butanone (DAB) reduces the amount of intracellular putrescine by 90%, preventing the cotransport of exogenous spermine. DAB-treated parasites present morphological changes, which are restored by adding exogenous putrescine into the culture medium. However, the effect of polyamines over the trichomonad proteomic profile is unknown. In this study, we used a proteomic approach to analyze the polyamine-depletion and restoration effect by exogenous putrescine on T. vaginalis proteome. In the presence of inhibitor DAB, we obtained 369 spots in polyamine-depleted condition and observed 499 spots in the normal culture media. With DAB treatment, the intensity of 43 spots was increased but was found to be reduced in 39 spots, as compared to normal conditions. Interestingly, in DAB-treated parasites restored with a medium with added exogenous putrescine, 472 spots were found, of which 33 were upregulated and 63 were downregulated in protein intensity. Some of these downregulated proteins in DAB-treated parasites are involved in several cellular pathways such as glycolysis, glycolytic fermentation, arginine dihydrolase pathway, redox homeostasis, host cell binding mediated by carbohydrate, chaperone function, and cytoskeletal remodeling. Interestingly, the intensity of some of the proteins was restored by adding exogenous putrescine. In conclusion, the presence of DAB altered the proteomic profile of T. vaginalis, resulting in a decrease in the intensity of 130 proteins and an increase in the intensity of 43 proteins that was restored by the addition of putrescine.

The 50kDa metalloproteinase TvMP50 is a zinc-mediated Trichomonas vaginalis virulence factor.

Trichomonas vaginalis is a protozoan parasite that can adapt to the trichomonicidal Zn2+ concentrations of the male urogenital tract microenvironment. This adaptation is mediated by molecular mechanisms, including proteinase expression, that are regulated by cations such as Zn2+. Herein, we characterized the previously identified 50kDa metalloproteinase aminopeptidase P (M24 family) member TvMP50 as a new Zn2+-mediated parasite virulence factor. Quantitative RT-PCR and indirect immunofluorescence assays corroborated the positive regulation of both mp50 gene expression and native TvMP50 protein overexpression in the cytoplasm and secretion products of parasites grown in the presence of Zn2+. Furthermore, this active metalloproteinase was characterized as a new virulence factor by assaying cytotoxicity toward prostatic DU145 cell monolayers as well as the inhibition of parasite and secreted soluble protein proteolytic activity in the 50kDa proteolytic region by the specific metalloproteinase inhibitor 1,10-phenanthroline and the chelating agents EDTA and EGTA. Parasite and secreted soluble protein cytotoxicity toward DU145 cells were reduced by treatment with an α-rTvMP50 polyclonal antibody. Our results show that the metalloproteinase TvMP50 is a new virulence factor modulated by Zn2+, which is present during male trichomoniasis, possibly explaining T. vaginalis survival even within the adverse conditions of the male urogenital microenvironment.

Putrescine-dependent re-localization of TvCP39, a cysteine proteinase involved in Trichomonas vaginalis cytotoxicity.

Polyamines are involved in the regulation of some Trichomonas vaginalis virulence factors such as the transcript, proteolytic activity, and cytotoxicity of TvCP65, a cysteine proteinase (CP) involved in the trichomonal cytotoxicity. In this work, we reported the putrescine effect on TvCP39, other CP that also participate in the trichomonal cytotoxicity. Parasites treated with 1,4-diamino-2-butanone (DAB) (an inhibitor of putrescine biosynthesis), diminished the amount and proteolytic activity of TvCP39 as compared with untreated parasites. Inhibition of putrescine biosynthesis also reduced ∼ 80% the tvcp39 mRNA levels according to RT-PCR and qRT-PCR assays. Additionally, actinomycin D-treatment showed that the tvcp39 mRNA half-life decreased in the absence of putrescine. However, this reduction was restored by exogenous putrescine addition, suggesting that putrescine is necessary for tvcp39 mRNA stability. TvCP39 was localized in the cytoplasm but, in DAB treated parasites transferred into exogenous putrescine culture media, TvCP39 was re-localized to the nucleus and nuclear periphery of trichomonads. Interestingly, the amount and proteolytic activity of TvCP39 was recovered as well as the tvcp39 mRNA levels were restored when putrescine exogenous was added to the DAB-treated parasites. In conclusion, our data show that putrescine regulate the TvCP39 expression, protein amount, proteolytic activity, and cellular localization.

TvMP50 is an immunogenic metalloproteinase during male trichomoniasis.

Trichomonas vaginalis, a human urogenital tract parasite, is capable of surviving in the male microenvironment, despite of the presence of Zn(2+). Concentrations > 1.6 mM of Zn(2+) have a trichomonacidal effect; however, in the presence of ≤1.6 mM Zn(2+), several trichomonad proteins are up- or down-regulated. Herein, we analyzed the proteome of a T. vaginalis male isolate (HGMN01) grown in the presence of Zn(2+) and found 32 protein spots that were immunorecognized by male trichomoniasis patient serum. Using mass spectrometry (MS), the proteins were identified and compared with 23 spots that were immunorecognized in the proteome of a female isolate using the same serum. Interestingly, we found a 50-kDa metallopeptidase (TvMP50). Unexpectedly, this proteinase was immunodetected by the serum of male trichomoniasis patients but not by the female patient serum or sera from healthy men and women. We analyzed the T. vaginalis genome and localized the mp50 gene in locus TVAG_403460. Using an RT-PCR assay, we amplified a 1320-bp mp50 mRNA transcript that was expressed in the presence of Zn(2+) in the HGMN01 and CNCD147 T. vaginalis isolates. According to a Western blot assay, native TvMP50 was differentially expressed in the presence of Zn(2+). The TvMP50 proteolytic activity increased in the presence of Zn(2+) in both isolates and was inhibited by EDTA but not by ptosyl-L-lysine chloromethyl ketone (TLCK), E64, leupeptin, or phenylmethane sulfonyl fluoride. Furthermore, the recombinant TvMP50 had proteolytic activity that was inhibited by EDTA. These data suggested that TvMP50 is immunogenic during male trichomoniasis, and Zn(2+) induces its expression.