Triosephosphate isomerase ofTrichomonas vaginalis(TvTIM) is a 27-kDa cytoplasmic protein encoded by two genes,tvtim1andtvtim2, that participates in glucose metabolism.TvTIM is also localized to the parasite surface. Thus, the goal of this study was to identify the novel functions of the surface-associatedTvTIM inT. vaginalisand to assess the effect of glucose as an environmental factor that regulates its expression and localization. Reverse transcription-PCR (RT-PCR) showed that thetvtimgenes were differentially expressed in response to glucose concentration.tvtim1was overexpressed under glucose-restricted (GR) conditions, whereastvtim2was overexpressed under glucose-rich, or high-glucose (HG), conditions. Western blot and indirect immunofluorescence assays also showed that glucose positively affected the amount and surface localization ofTvTIM inT. vaginalis. Affinity ligand assays demonstrated that the recombinantTvTIM1 andTvTIM2 proteins bound to laminin (Lm) and fibronectin (Fn) but not to plasminogen. Moreover, higher levels of adherence to Lm and Fn were detected in parasites grown under HG conditions than in those grown under GR conditions. Furthermore, pretreatment of trichomonads with an anti-TvTIMr polyclonal antibody or pretreatment of Lm- or Fn-coated wells with both recombinant proteins (TvTIM1r andTvTIM2r) specifically reduced the binding of live parasites to Lm and Fn in a concentration-dependent manner. Moreover,T. vaginaliswas exposed to different glucose concentrations during vaginal infection of women with trichomoniasis. Our data indicate thatTvTIM is a surface-associated protein under HG conditions that mediates specific binding to Lm and Fn as a novel virulence factor ofT. vaginalis.

We focus on the iron response ofTrichomonas vaginalisto gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes.

The goal of this paper was to characterize aTrichomonas vaginaliscysteine proteinase (CP) legumain-1 (TvLEGU-1) and determine its potential role as a virulence factor duringT. vaginalisinfection. A 30-kDa band, which migrates in three protein spots (pI~6.3, ~6.5, and ~6.7) with a different type and level of phosphorylation, was identified as TvLEGU-1 by one- and two-dimensional Western blot (WB) assays, using a protease-rich trichomonad extract and polyclonal antibodies produced against the recombinant TvLEGU-1 (anti-TvLEGU-1r). Its identification was confirmed by mass spectrometry. Immunofluorescence, cell binding, and WB assays showed that TvLEGU-1 is upregulated by iron at the protein level, localized on the trichomonad surface and in lysosomes and Golgi complex, bound to the surface of HeLa cells, and was found in vaginal secretions. Additionally, the IgG and Fab fractions of the anti-TvLEGU-1r antibody inhibited trichomonal cytoadherence up to 45%. Moreover, the Aza-Peptidyl Michael Acceptor that inhibited legumain proteolytic activity in live parasites also reduced levels of trichomonal cytoadherence up to 80%. In conclusion, our data show that the proteolytic activity of TvLEGU-1 is necessary for trichomonal adherence. Thus, TvLEGU-1 is a novel virulence factor upregulated by iron. This is the first report that a legumain-like CP plays a role in a pathogen cytoadherence.