Specific and reversible inhibition of entamoeba histolytica cysteine proteinase activities by Zn²+: Implications for adhersion and cell damage1

RESUMO

Background. Cysteine-proteinases are thought to play an important role in E. histolytica pathogenicity. Although effective blockage of proteolytic activities can be obtained with sereveral known inhibitors, the high cellular toxicity of most of the inhibitors precludes experimentation with live cells or animal models. Specific cysteine-proteinase inhibitors that could be utilized in studies of virulence are of great need in the field of amebiasis. Methods. Cysteine-proteinase activities were determined in trophozoit lysates by azocasein degradation and after PAGE and gelatin zymograms. Inhibition of the activities was assessed in the presence of 0.01-2.5 mM concentrations of fivalent cations of the IIB and VIII series such as Zn, Cd, Hg, Ni, and Co. Reversibility was induced with 25 mM L-cysteine or 50 mM L-histidine and by metal chelation with 5 mM phenantroline. The inhibitory effect of ZnCI2 was tested with live cells in fibronectin-biding and cytotoxicity assays. Results. ZnCI2 specifically inhibited cysteine-proteinase activities in trophozoite lysates in a concentration-dependent manner. Additionally, 1.0-2.5 mM ZnCI2 bloked proteolysis in more than 70 percent. This inhibition was completely reverted by L-cysteine, L-histidine, or phenantroline. Similar results were obtained by analyzing indivual cysteine-proteinase activities separated in gelatin zymograms. At these concentrations, ZnCI2 significanty interfered with trophozoit adhesion, thus making amebas deficient in substrate degradation and cell damage. Conclusions. ZnCI2 is effective inhibitor of amebic cysteine-proteinases. Its low toxicity at relative high concentrations, high solubility, and low cost make it adequate for live cell experimentation and animal models of amebic virulence