CYSTEINE PROTEASE INHIBITORS ALTER GOLGI-COMPLEX ULTRASTRUCTURE AND FUNCTION IN TRYPANOSOMA-CRUZI

Cruzain, the major cysteine protease of the protozoan parasite Trypano soma cruzi, is a target of rational drug design for chemotherapy of Ch agas' disease. The precise biological role of cruzain in the parasite life cycle and the mechanism involved in the trypanocidal effect of cy steine protease inhibitors are still unclear, Here we report biologica l and ultrastructural alterations caused by cysteine protease inhibito rs in T. cruzi epimastigotes. Cruzain, a glycoprotein that transits th e Golgi-endosomal pathway, localized to pre-lysosomes/lysosomes in the posterior end of untreated epimastigotes by fluorescent microscopy ut ilizing either a biotinylated cysteine protease inhibitor to tag the a ctive site, or a specific anti-cruzain antibody. Radiolabeled or bioti nylated cysteine protease inhibitors bound exclusively to cruzain in i ntact epimastigotes confirming that cruzain is accessible to, and is t argeted by the inhibitors. Treatment of T. cruzi epimastigotes with sp ecific cysteine protease inhibitors arrested growth, altered the intra cellular localization of cruzain, and induced major alterations in the Golgi complex. Following treatment, cruzain accumulated in peripheral dilations of Golgi cisternae. There was a concomitant 70% reduction i n gold-labeled cruzain transported to lysosomes. Cisternae abnormaliti es in the Golgi compartment were followed by distention of ER and nucl ear membranes, Brefeldin A increased the number and size of cisternae in epimastigotes. Pre-treatment of epimastigotes with cysteine proteas e inhibitors followed by exposure to brefeldin A induced a more rapid appearance of the cysteine protease inhibitor-induced Golgi alteration s. Our results suggest that cysteine protease inhibitors prevent the n ormal autocatalytic processing and trafficking of cruzain within the G olgi apparatus. Accumulation of cruzain may decrease mobility of Golgi membranes and result in peripheral distention of cisternae. These maj or alterations of the Golgi complex parallel the death of T. cruzi epi mastigotes.