María Elizbeth Alvarez-Sánchez

Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage

Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.

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 Zn2+. Concentrations > 1.6 mm of Zn2+ have a trichomonacidal effect; however, in the presence of ≤1.6 mm Zn2+, 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 Zn2+ 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 Zn2+ in the HGMN01 and CNCD147 T. vaginalis isolates. According to a Western blot assay, native TvMP50 was differentially expressed in the presence of Zn2+. The TvMP50 proteolytic activity increased in the presence of Zn2+ 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 Zn2+ induces its expression.

Identification of two novel Trichomonas vaginalis eif-5a genes.

The eukaryotic translation initiation factor 5A (eIF-5A) is highly conserved and is the only protein that is known to contain the unique and essential amino acid residue hypusine. Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. In this study, we identified two novel eukaryotic translation initiation factor 5A (eIF-5A) genes in Trichomonas vaginalis. The tveif-5a1 and tveif-5a2 putative genes were localized in different contigs, both containing ORFs encoding proteins of 168 amino acids that share high sequence identity with eIF-5A sequences from other eukaryotic organisms. A phylogenetic tree constructed with TveIF-5A1 and TveIF-5A2 from T. vaginalis and 13 other eIF-5A sequences of eukaryotic and archaebacterial origin revealed that both trichomonal TveIF-5As show the highest degree of similarity to bacteria. Using an anti-TveIF-5A antibody, we detected two protein bands and spots of 19 and 20kDa with isoelectric points (pI) of 5.2 and 5.5, respectively, by one and two-dimensional Western blot assays. In addition, we used reverse transcription polymerase chain reaction (RT-PCR) to demonstrate that both of these tveif-5a genes are expressed in T. vaginalis. Immunofluorescence assays showed that the TveIF-5A protein was dispersed throughout the parasite cytoplasm. In conclusion, T. vaginalis has two eif-5a genes, and both genes are expressed as highly conserved proteins of 19kDa, which are localized in the cytoplasm of this parasite.

The identification of a VDAC-like protein involved in the interaction of Babesia bigemina sexual stages with Rhipicephalus microplus midgut cells

We investigated the interaction of Rhipicephalus microplus midgut cells with Babesia bigemina sexual stages using a proteomic approach. A polypeptide from the R. microplus midgut that binds to proteins from B. bigemina sexual stages was identified and sequenced. Combining 2D overlay and tandem mass spectrometry (MS/MS) techniques, we determined that this polypeptide corresponds to a mitochondrial voltage-dependent anion-selective channel (VDAC). The vdac gene encoding the sequenced polypeptide was identified and sequenced. This is the first report of a VDAC-like protein in R. microplus, and a possible role for this protein in the B. bigemina infection process is suggested.

Putrescine is required for the expression of eif-5a in Trichomonas vaginalis

Recently, we found that Trichomonas vaginalis contains a eukaryotic translation initiation factor 5A (TveIF-5A) with unknown function in this parasite. eIF-5A is the only cellular protein dependent of polyamines to form a hypusine residue, an unusual basic amino acid that is post-translationally formed by modification of a single specific lysine residue in an eIF-5A precursor protein. The purpose of this study was to determine the effect of a putrescine analogue, 1,4-diamino-2-butanone (DAB), on tveif-5a mRNA and TveIF-5A protein expression. TveIF-5A protein expression was reduced by inhibition of putrescine biosynthesis, and tveif-5a mRNA levels were reduced ∼90%, as shown by western blot and immunofluorescence assays. Cycloheximide treatment reduced the amount of mature TveIF-5A protein at 4h and decreased the tveif-5a transcript level at 2h, according to western blot, RT-PCR and qRT-PCR analyses. Actinomycin D treatment showed that the tveif-5a mRNA had half-life of ∼2.5h in DAB-treated parasites. The half-life of tveif-5a mRNA was ∼4.5h under exogenous putrescine conditions. These results suggest that putrescine is required for tveif-5a mRNA stability, and it is necessary for the expression, stability and maturation of TveIF-5A protein.

The 50 kDa 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.

Genome-wide identification, in silico characterization and expression analysis of ZIP-like genes from Trichomonas vaginalis in response to Zinc and Iron

Trace elements such as Zinc and Iron are essential components of metalloproteins and serve as cofactors or structural elements for enzymes involved in several important biological processes in almost all organisms. Because either excess or insufficient levels of Zn and Fe can be harmful for the cells, the homeostatic levels of these trace minerals must be tightly regulated. The Zinc regulated transporter, Iron regulated transporter-like Proteins (ZIP) comprise a diverse family, with several paralogues in diverse organisms and are considered essential for the Zn and Fe uptake and homeostasis. Zn and Fe has been shown to regulate expression of proteins involved in metabolism and pathogenicity mechanisms in the protozoan pathogen Trichomonas vaginalis, in contrast high concentrations of these elements were also found to be toxic for T. vaginalis trophozoites. Nevertheless, Zn and Fe uptake and homeostasis mechanisms is not yet clear in this parasite. We performed a genome-wide analysis and localized the 8 members of the ZIP gene family in T. vaginalis (TvZIP1-8). The bioinformatic programs predicted that the TvZIP proteins are highly conserved and show similar properties to the reported in other ZIP orthologues. The expression patterns of TvZIP1, 3, 5 and 7 were diminished in presence of Zinc, while the rest of the TvZIP genes showed an unchanged profile in this condition. In addition, TvZIP2 and TvZIP4 showed a differential expression pattern in trophozoites growth under different Iron conditions. These results suggest that TvZIP genes encode membrane transporters that may be responsible for the Zn and Fe acquisition in T. vaginalis.

BmVDAC upregulation in the midgut of Rhipicephalus microplus, during infection with Babesia bigemina.

The molecular mechanisms involved during the infection of Rhipicephalus microplus midgut cells by Babesia bigemina are of great relevance and currently unknown. In a previous study, we found a voltage-dependent anion channel (VDAC)-like protein (BmVDAC) that may participate during parasite invasion of midgut cells. In this work, we investigated BmVDAC expression at both mRNA and protein levels and examined BmVDAC localization in midgut cells of ticks infected with B. bigemina at different times post-repletion. Based on the RT-PCR results, Bmvdac expression levels were significantly higher in infected ticks compared to uninfected ones, reaching their highest values at 24h post-repletion (p<0.0001). Similar results were obtained at the protein level (p<0.0001). Interestingly, BmVDAC immunolocalization showed that there was an important differential expression and redistribution of BmVDAC protein between the midgut cells of infected and uninfected ticks, which was more evident 24h post-repletion of infected ticks. This is the first report of BmVDAC upregulation and immunolocalization in R. microplus midgut cells during B. bigemina infection. Further studies regarding the function of BmVDAC during the infection may provide new insights into the molecular mechanisms between B. bigemina and its tick vector and could result in its use as an anti-tick and transmission-blocking vaccine candidate.

Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation

BackgroundBovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future.ResultsTo isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro.ConclusionBabesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.