In general we observed that
In general, we observed that the enolase gene with highest mRNA levels was TVAG_043500, under all three iron concentration conditions. mRNA levels for this gene are very similar in normal and iron rich cultures (73.9 and 78.1 fold change, respectively), and higher in iron depleted cultures (118.3 fold change). TVAG_358110 occupies the second place of mRNA levels at normal and high concentrations of iron (4.2 and 8.1 fold change respectively), whereas the Pyrimethamine is less abundant in low iron concentration conditions (1.3 fold change). On the opposite, Trichomonas vaginalis grown at normal and high iron concentration contain very low levels of genes corresponding to TVAG_263740, TVAG_170370, TVAG_282090, TVAG_464170 and TVAG_329460.
Discussion Enolase from Trichomonas vaginalis is a moonlighting protein that apart from the canonic participation in glycolysis, also acts as a plasminogen receptor on the surface of the parasite, and in consequence, it has been described as a new surface-associated virulence factor. Here we performed a molecular characterization of each of the nine genes encoding for this protein, as well as their expression patterns. All nine contigs annotated as enolase in TrichDB (www.trichdb.org) are independent form each other, since they all are in different contigs. Seven enolases (encoded by TVAG_464170, TVAG_043500, TVAG_329460, TVAG_358110, TVAG_263740, TVAG_487600 and TVAG_282090) are competent of performing enzymatic activity, since they harbor the majority of the aminoacid residues of the catalytic moiety, as well as divalent cation binding site residues reported for enolases from other organisms [7,34,36]. Interestingly the same seven enolases are also prone to bind plasminogen. These results argue against the hypothesis that some members of the enolase family could bind plasminogen and others could have enzyme activity. In addition, the same group also harbor host derived exRNA-binding motifs. To confirm the functional plasticity of these enolases further studies are needed. We could detect the amplification of all enolase genes except for TVAG_487600, although we tested several experimental conditions. Furthermore, we observed that for most enolase genes mRNA levels decrease with iron concentration, the highest exception is TVAG_358110. On the contrary, other authors have reported down regulation in iron depleted conditions (TVAG_464170, −2.65 fold change and TVAG_043500, −14.3 fold change) . In comparison, the highest number of ESTs reports is for TVAG_464170, followed by TVAG_043500, TVAG_358110 and TVAG_329460 in third and fourth place, respectively. Nevertheless, all condition tested in ESTs reports are with different strains and culture conditions to those used in this study. Respect to the proteomics approach, three protein spots could be resolved and identified by antibody anti-enolase. Only one enolase could be unequivocally detected (TVAG_464170) in all iron concentration conditions. The other two spots provided the same peptides which match with two enolases (TVAG_043500 and TVAG_329460), which might be differentiated by their theoretical isoelectric points. Although TVAG_358110 show high mRNA levels at normal and high iron concentration conditions, it could not be detected by mass spectrometry analysis. Cis-regulatory elements of this gene do not show any evident difference in comparison with those genes that could be detected by the proteomics approach. In particular, finding the product of TVAG_329460 is interesting since it has been encountered on the cell surface of B7RC2 strain (PA strain, ATCC 50167)  , and additionally it has been probed to perform the moonlighting activity as plasminogen receptor on the cell surface of T016 strain , and therefore, described as a new surface-associated virulence factor. Although, as it has been mentioned, seven enolases are prone to perform plasminogen-binding activity, and in other T. vaginalis strains TVAG_464170 and TVAG_358110 have been detected in cytoadherence   conditions, as well as in the surface proteome .