A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity

The goal of this study was to demonstrate the participation in cellular dam age of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa ( CP65). By two dimensional gelatin-gel electrophoresis of trichomonad protei ns we detected four spots with proteolytic activity on the 65 kDa region, b ut only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluo rescence, rabbit antibodies against this proteinase localized the CP65 on t he plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of pa rasites with the specific anti-CP65 antibody reduced trichomonal cytotoxici ty to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3 -carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) a brogated the proteinase activity and reduced cytotoxicity levels of T: vagi nalis in cell culture monolayers, indicating that the trichomonad CP65 is a cysteine proteinase. Activity of the CP65 proteinase was optimal at pH 5.5 and 37 degrees C, conditions similar to those of patients with trichomonos is. Also, this proteinase degraded some of the proteins found in the vagina , i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Final ly, immunoprecipitation assays showed that sera and vaginal washes from tri chomonosis patient possess anti-CP65 antibodies. In conclusion, results pre sented in this work demonstrate that the CP65 is a surface cysteine protein ase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a vir ulence factor. It is immunogenic during human infection and degrades some e xtracellular matrix proteins, i.e. collagen IV and fibronectin. (C) 2000 Ac ademic Press.