In silico analysis of putative metal response elements (MREs) in the zinc-responsive genes from Trichomonas vaginalis and the identification of novel palindromic MRE-like motif.

Zinc is an essential micronutrient that plays an important role as a co-factor to several proteins, including zinc-responsive transcription factors. Trichomonas vaginalis is able to survive in the presence of high zinc concentrations in the male urogenital tract. Several genes in T. vaginalis have been shown to respond to changes in zinc concentrations, however, the zinc-dependent mechanism remains undetermined. Recently, we identified in T. vaginalis the zinc finger protein, TvZNF1, which is an ortholog of the mammal metal transcription factor (MTF1). We searched for several of the zinc-responsive genes in T. vaginalis to determine whether if they contain metal response elements (MRE), cis-acting DNA elements that specifically bind MTF1. Six highly conserved over-represented sequence motifs (TvMREs), which share similarity with other eukaryotic MREs, were identified in the zinc-responsive genes in T. vaginalis. We also demonstrated that some of the TvMREs assemble as divalent complexes either as two closely spaced TvMREs or as two overlapping TvMREs forming a palindromic-like sequence: TGCC(N3)GGCA. Electrophoretic mobility shift assays were used to detect the zinc-dependent binding of TvZNF1 and nuclear proteins from T. vaginalis to this specific palindromic motif. Our results support a novel mechanism used by T. vaginalis for the transcriptional regulation of associated zinc-responsive genes through a MTF1/MRE-like system.

Trichomonas vaginalis metalloproteinase TvMP50 is a monomeric Aminopeptidase P-like enzyme.

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.

TvZNF1 is a C2H2 zinc finger protein of Trichomonas vaginalis.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

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.

Polyamine Transport and Synthesis in Trichomonas vaginalis: Potential Therapeutic Targets.

Polyamines are essential for many biological processes in all organisms. Here we show a current landscape of studies and strategies implemented for the study of polyamine metabolism, as well as molecular aspects that implicate the role of key enzymes, transport proteins, inhibitors, and the study of novel molecules as potential therapeutic targets. This review focused on the synthesis, interconversion and function of these molecules in Trichomonas vaginalis, a common sexually transmitted parasite of humans.

Bifunctional activity of deoxyhypusine synthase/hydroxylase from Trichomonas vaginalis.

The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ((3)H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 µM. The rTvDHS activity was inhibited by the spermidine analog, N″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity.

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.