Negative iron regulation of the CP65 cysteine proteinase cytotoxicity in Trichomonas vaginalis.

Several cysteine proteinases (CPs) participate in the virulence of Trichomonas vaginalis. One of them is a 65kDa CP, CP65, involved in cytotoxicity. The aim of this work was to investigate the effect of iron on the trichomonal CP65-dependent cytotoxicity using parasites grown under distinct iron concentrations. Cytotoxicity and cell-binding assays, and zymograms were performed. At the highest iron concentration (250 microM), parasites exhibited the lowest levels of cytotoxicity and less CP65 proteolytic activity. Other cations in the culture medium did not affect the trichomonal CP65-dependent cytotoxicity as iron did. Another four trichomonad fresh isolates presented similar iron negative effect over cytotoxicity. Western blot and RT-PCR experiments also showed reduction in the amount of protein and transcript of CP65 in trichomonads grown under iron-rich conditions, as compared with parasites grown in normal and iron-depleted media. Indirect immunofluorescence using the anti-CP65 antibody showed that parasites grown in iron-rich medium expressed less CP65 than those grown in normal and iron-depleted media. Cytotoxicity inhibition experiments with the anti-CP65 antibody confirmed the iron negative effect over the CP65-dependent cytotoxicity. In conclusion, our data show that iron specifically down-regulates proteolytic activity, expression, and transcription of CP65, negatively affecting trichomonal cytotoxicity in vitro.

A Trichomonas vaginalis 120 kDa protein with identity to hydrogenosome pyruvate:ferredoxin oxidoreductase is a surface adhesin induced by iron.

Trichomonas vaginalis, a human sexually transmitted protozoan, relies on adherence to the vaginal epithelium for colonization and maintenance of infection in the host. Thus, adherence molecules play a fundamental role in the trichomonal infection. Here, we show the identification and characterization of a 120 kDa surface glycoprotein (AP120) induced by iron, which participates in cytoadherence. AP120 is synthesized by the parasite when grown in 250 microM iron medium. Antibodies to AP120 and the electro-eluted AP120 inhibited parasite adherence in a concentration-dependent manner, demonstrating its participation in cytoadherence. In addition, a protein of 130 kDa was detected on the surface of HeLa cells as the putative receptor for AP120. By peptide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the AP120 adhesin showed homology with a hydrogenosomal enzyme, the pyruvate:ferredoxin oxidoreductase (PFO) encoded by the pfoa gene. This homology was confirmed by immunoblot and indirect immunofluorescence assays with an antibody to the carboxy-terminus region of the Entamoeba histolytica PFO. Reverse transcription polymerase chain reaction (RT-PCR) assays showed that a pfoa-like gene was better transcribed in trichomonads grown in iron-rich medium. In conclusion, the homology of AP120 to PFO suggests that this novel adhesin induced by iron could be an example of moonlighting protein in T. vaginalis.