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.

SUMMARY: The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report thatTrichomonas vaginaliscontains 2 fully functionaltpigenes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located inα-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed thattpi2transcript is approximately 3·3-fold more abundant thantpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement anEscherichia colistrain carrying a deletion of its endogenoustpigene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM fromSaccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. AsT. vaginalisdepends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicatedtpicopy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.