Upregulation of the secretory pathway in cysteine protease inhibitor-resistant Trypanosoma cruzi

A novel chemotherapy in development for Chagas' disease targets cruzain, th e major cysteine protease of Trypanosoma cruzi, Peptidomimetic inhibitors d isrupt the intracellular cycle of the parasite and rescue animals from a le thal infection, Inhibitor killing of parasites results from interruption of autocatalytic cruzain processing and transport to lysosomes, and massive a ccumulation of precursor protein in the Golgi complex. To further understan d the mechanisms of protease processing and transport in this primitive euk aryote, and uncover potential mechanisms for resistance to these drugs, we generated cysteine-proteaae inhibitor (CPI)-resistant epimastigotes in vitr o and investigated the mechanisms involved at the biochemical and structura l levels. Resistance to 20-fold the lethal CPI concentration, achieved afte r a year of gradual drug increase, was accompanied by a modest decrease in growth rate, A marked increase in the number of vesicles trafficking from t he Golgi complex to the flagellar pocket occurs in resistant cells. No matu re protease reaches lysosomes though accumulation of endocytosed gold parti cles in lysosomes appears to be normal. Higher molecular mass cruzain speci es, consistent with complexes of cruzain precursors and inhibitor, are secr eted by CPI-resistant parasites into the culture supernatant, Release of th ese cruzain precursors may be facilitated by an enhanced acidification of t rans-Golgi cisternae in resistant parasites. The pH within Golgi cisternae is higher in control epimastigotes and most mature cruzain is lysosomal, Cr uzain activity is negligible in CPI-resistant epimastigote extracts compare d to the parental clone. Activity is restored following withdrawal of the i nhibitor. No cross-resistance to the therapeutic drugs nifurtimox and benzn idazole occurred and, conversely, parasites resistant to these drugs were s ensitive to CPI, Protease inhibitors are thus potential therapeutical alter natives in cases of nifurtimox/benznidazole resistance. Cumulatively, these results suggest that CPI-resistance induces upregulation of Golgi complex function and post-Golgi secretory pathway, and release of precursors before the enzyme reaches its site of biologic activity.