Cloning of a cysteine protease required for the molting of Onchocerca volvulus third stage larvae

We have investigated the involvement of a cysteine protease in the development of Onchocerca volvulus fourth stage larvae (L4) by testing the effect of cysteine protease inhibitors on the survival of third stage larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluoromethylketones, viability of either L3 or L4 was not affected. However, the inhibitors reduced the number of L3 that molted to L4 in vitro in a time- and dose-dependent manner. Molting was completely inhibited in the presence of 50-250 microM inhibitor. Ultrastructural examination of L3 that did not molt in the presence of inhibitors indicated that new L4 cuticle was synthesized, but there was no separation between the L3 and the L4 cuticles. The endogenous cysteine protease was detected in molting larvae after binding to labeled inhibitors, and by antibodies directed against a recombinant O. volvulus L3 cysteine protease that was cloned and expressed. The enzyme was detected in cuticle regions where the separation between the cuticles occurs in molting larvae. These studies suggest that molting and successful development of L4 depends on the expression and release of a cysteine protease.