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Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage.

The cysteine proteinase (CP) of 65kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67-76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (alpha-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.