The TvLEGU-1, a Legumain-Like Cysteine Proteinase, Plays a Key Role in Trichomonas vaginalis Cytoadherence
Figure 8
Effect of distinct CP inhibitors in trichomonal cytoadherence ((A) and (B)) and proteolytic activity of live parasites ((C) and (D)). (A) Fluorescence microscopy of a representative cytoadherence inhibition assay (over live HeLa cell monolayers) of fluorescence-labeled parasites pretreated with different CP inhibitors. Panels a and b show parasites without treatment (100% adherence). Panels c and d correspond to parasites treated with 50 μM legumain inhibitor Aza-Peptide Michael Acceptor (Mu-Ala-Ala-AAsn-CH=CH-CON). Panels e and f show parasites treated with 1 mM TLCK. Panels g and h, parasites treated with 0.2 mM Leupeptin (Leup). Panels i and j, parasites treated with 0.18 mM E-64. (B) Data from the fluorescent parasites of the cytoadherence inhibition assay show the percentage of T. vaginalis bound to HeLa cell monolayers in the absence (0, used as a control) or presence of distinct concentrations of CP inhibitors described in (A). Each bar is the mean of the percentage of triplicate samples; error bars represent the standard deviations of two experiments in triplicate with similar results. * is the significance of the difference between the control and the distinct treatments. (C) Proteolytic activity of live trichomonads over legumain substrate (Cbz-Ala-Ala-AAsn-AMC). Live parasites were incubated with the same inhibitors previously described in (A), and the released fluorescence from the legumain substrate was measured in a fluorometer. Each bar is the mean of the percentage of triplicate samples; error bars represent the standard deviations of three experiments in triplicate with similar results. * is the significance of the difference between the control and the distinct treatments. (D) Proteolytic activity of live trichomonads over papain substrate (Z-Phe-Arg-AMC). Live parasites were incubated with the same inhibitors previously described in (A), and the released fluorescence from the papain substrate was measured in a fluorometer. Each bar is the mean of the percentage of triplicate samples; error bars represent the standard deviations of three experiments in triplicate with similar results. * is the significance of the difference between the control and the distinct treatments.
