The Non-Canonical 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.

Trichomonas vaginalis is one of the most common sexually transmitted parasites in humans. This protozoan has high iron requirements for growth, metabolism, and virulence. However, iron concentrations also differentially modulate T. vaginalis gene expression as in the genes encoding cysteine proteinases TvCP4 and TvCP12. Our goal was to identify the regulatory mechanism mediating the upregulation of tvcp12 under iron-restricted (IR) conditions. Here, we showed by RT-PCR, Western blot, and immunocytochemistry assays that IR conditions increase mRNA stability and amount of TvCP12. RNA electrophoretic mobility shift assay (REMSA), UV cross-linking, and competition assays demonstrated that a non-canonical iron-responsive element (IRE)-like structure at the 3'-untranslated region of the tvcp12 transcript (IRE-tvcp12) specifically binds to human iron regulatory proteins (IRPs) and to atypical RNA-binding cytoplasmic proteins from IR trichomonads, such as HSP70 and α-Actinin 3. These data were confirmed by REMSA supershift and Northwestern blot assays. Thus, our findings show that a positive gene expression regulation under IR conditions occurs at the posttranscriptional level possibly through RNA-protein interactions between atypical RNA-binding proteins and non-canonical IRE-like structures at the 3'-UTR of the transcript by a parallel mechanism to the mammalian IRE/IRP system that can be applied to other iron-regulated genes of T. vaginalis.

Cloning and Recombinant Expression of Elongation Factor-1α of Leishmania mexicana.

Leishmania mexicana is an intracellular parasite that causes cutaneous leishmaniasis (CL) in some countries, including Mexico. Recently, we identified the elongation factor-1α (EF-1α) of L. mexicana by immunoproteomic analysis. In Leishmania donovani, this molecule has been reported as a virulence factor involved in downregulation of macrophages by no-canonical function when EF-1α interacts with protein tyrosine phosphatase-1 (SHP-1). However, in L. mexicana the key role of EF-1α in host-parasite relationship has not been elucidated, by this reason we started with cloning and recombinant expression of this antigen. A sequence of 1350 bp corresponding to EF-1α (EF-Lm) full-length gene was amplified from genomic DNA of L. mexicana (GenBank: MG256973); this gene contains only one nucleotide change: C464T, compared with L. mexicana reference sequence (GenBank: FR799570.1). The gene cloned (EF-Lm) codes for a protein of 449 residues. It was expressed in Escherichia coli and purified as 63 kDa sumo-fusion protein, which was recognized in the sera of patients diagnosed with CL. Our results show that EF-Lm is immunogenic during infection, and the rEF-Lm could be used for further analyses in the host-parasite relationship.

Immunoproteomic Identification of p29 Antigen as the Elongation Factor-1α of Leishmania mexicana.

Previously, we identified five Leishmania mexicana antigens reacting with antibodies from cutaneous leishmaniasis patients, designated on the basis of their molecular weights as p26 (pI 7.8), p27 (pI 8.1), p28 (pI 8.6), p29 (pI 8.5), and p31 (pI 9.0). Among these antigens, p29 was most strongly recognized by the antibodies. Thereafter, p29 was identified as elongation factor-1α (EF-1α) of Leishmania mexicana through mass spectrometry analysis and western blot using a commercial antibody that reacted with EF-1α from different species. Our results showed that the p29 antigen of Leishmania mexicana is EF-1α.

tvcp12: a novel Trichomonas vaginalis cathepsin L-like cysteine proteinase-encoding gene.

Trichomonas vaginalis is the causative agent of trichomoniasis, one of the most common sexually transmitted diseases in humans. This protozoan has multiple proteinases that are mainly of the cysteine proteinase (CP) type, some of which are known to be involved in the parasite's virulence. Here, a novel T. vaginalis CP-encoding gene, tvcp12, was identified and characterized. tvcp12 is 948 bp long and encodes a predicted 34.4 kDa protein that has the characteristics of the papain-like CP family. TvCP12 does not appear to have a signal peptide, suggesting that this is a cytoplasmic CP. By Southern blot assays, the tvcp12 gene was found as a single copy in the T. vaginalis genome. Remarkably, Northern blot experiments showed a single transcript band of approximately 1.3 kb in the mRNA obtained from parasites grown in low iron conditions and no transcript was observed in the mRNA from parasites grown in high iron conditions. By RT-PCR assays, a 270 bp band was amplified from the cDNA of parasites grown in low iron medium, which was very faint when cDNA from parasites grown in high iron conditions was used. Transcripts of the 3' region obtained in both iron conditions presented differences in their poly(A) tail length. These data suggest that tvcp12 is another gene that is negatively regulated by iron and that the length of the poly(A) tail may be one of the factors involved in the iron-modulated protein expression.