A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity.
The goal of this study was to demonstrate the participation in cellular damage of a Trichomonas vaginalis proteinase with a molecular mass of 65 kDa (CP65). By two dimensional gelatin-gel electrophoresis of trichomonad proteins we detected four spots with proteolytic activity on the 65 kDa region, but only one, pI 7.2, binds to the HeLa cell surface. By indirect immunofluorescence, rabbit antibodies against this proteinase localized the CP65 on the plasma membrane and in the cytoplasm of T. vaginalis. Pretreatment of parasites with the specific anti-CP65 antibody reduced trichomonal cytotoxicity to HeLa cell monolayers. The specific cysteine proteinase inhibitor, L-3-carboxy-2, 3-trans-epoxypropionyl-leucylamido (4-guanidino) butane (E64) abrogated the proteinase activity and reduced cytotoxicity levels of T. vaginalis 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 trichomonosis. Also, this proteinase degraded some of the proteins found in the vagina, i.e. collagen IV and fibronectin, but not laminin-1 or haemoglobin. Finally, immunoprecipitation assays showed that sera and vaginal washes from trichomonosis patient possess anti-CP65 antibodies. In conclusion, results presented in this work demonstrate that the CP65 is a surface cysteine proteinase involved in T. vaginalis cytotoxicity to HeLa cell monolayers, as a virulence factor. It is immunogenic during human infection and degrades some extracellular matrix proteins, i.e. collagen IV and fibronectin.
Alvarez-Sánchez
M E
ME
Departamento de Patología Experimental, México, 07360, D.F. México.
Avila-González
L
L
Becerril-García
C
C
Fattel-Facenda
L V
LV
Ortega-López
J
J
Arroyo
R
R
D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D016923:Cell Death; D002462:Cell Membrane / Q000201:enzymology; D003094:Collagen / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000276:immunology / Q000378:metabolism*; D004795:Enzyme Stability; D005260:Female; D005353:Fibronectins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011817:Rabbits; D014247:Trichomonas Vaginitis / Q000276:immunology / Q000469:parasitology*; D014246:Trichomonas vaginalis / Q000201:enzymology* / Q000472:pathogenicity*; D014774:Virulence
D000913:Antibodies, Protozoan; D005353:Fibronectins; D003094:Collagen; D003546:Cysteine Endopeptidases