Characterization of the Trichomonas vaginalis AP65 adhesin and effects of polyamine depletion on cytoadherence and cytotoxicity

Trichomonas vaginalis colonizes the human urogenital tract and is the causative agent of trichomonosis, the most common sexually transmitted infection (STI) in women. Five surface proteins (AP120, AP65, AP51, AP33, and AP23) mediate adherence to vaginal epithelial cells (VECs) (Alderete and Garza, 1988; Arroyo et al., 1993; Garcia et al., 2003; Arroyo et al., 2005; Kucknoor et al., 2005). These proteins are also found in the hydrogenosome organelles as metabolic enzymes (Muller, 1993). Three adhesin proteins were co-expressed and co-localized on the cell surface under high-iron conditions. Furthermore, the AP65 adhesin epitopes seen by rabbit anti-AP65 serum that blocks adherence and detects surface proteins were identified. In addition, contact of trichomonads with VECs increased the amount of surface-expressed adhesins. Lab created MR100, a drug-resistant isolate, was found to have reduced numbers of hydrogenosomes and little or no detectable enzymes. Therefore, not surprisingly, MR100 organisms were non-adherent. These results confirm an important role for iron and contact in modulating synthesis and surface expression of T. vaginalis adhesins. As AP65 is a secreted protein, we also demonstrated anchorless, surface-association of AP65 to the membrane. The binding epitope interacting with both organisms and VECs was identified by testing overlapping recombinant subclones of AP65. It was found that a full N-terminal domain of AP65 was required for binding to parasites and VECs. Further, polyamine depletion was correlated with increased levels of adherence without affecting adhesins. Importantly trichomonal polyamine depletion resulted in the abolishment of contact-dependent cytotoxicity mediated by a specific cysteine proteinase, called CP65, which was concomitantly down-regulated in expression. Overall my work found the following: (1) compartmentalization of adhesins outside the hydrogenosomes is modulated by iron; (2) AP65 is a prominent adhesin mediating adherence to VECs; (3) secreted AP65 re-associates with trichomonal surfaces by interacting with a membrane proteinaceous structure; (4) the intact N-terminal region of AP65 is essential for adhesin interacting with both organisms and VECs; (5) polyamine depletion increases cytoadherence while abolishing contact-dependent host killing; and (6) there is a relationship between polyamine metabolism and gene expression of a cytotoxic cysteine proteinase, and possibly other trichomonad genes.