CLONING OF A CYSTEINE PROTEASE REQUIRED FOR THE MOLTING OF ONCHOCERCA-VOLVULUS 3RD STAGE LARVAE

We have investigated the involvement of a cysteine protease in the dev elopment of Onchocerca volvulus fourth stage larvae (L4) by testing th e effect of cysteine protease inhibitors on the survival of third stag e larvae (L3), and the molting of L3 to L4 in vitro. When larvae were cultured in the presence of specific inhibitors, the peptidyl monofluo romethylketones, viability of either L3 or L4 was not affected, Howeve r, 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 mu M 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 th e L3 and the L4 cuticles, The endogenous cysteine protease was detecte d in molting larvae after binding to labeled inhibitors, and by antibo dies directed against a recombinant O. volvulus L3 cysteine protease t hat was cloned and expressed. The enzyme was detected in cuticle regio ns where the separation between the cuticles occurs in molting larvae, These studies suggest that molting and successful development of L4 d epends on the expression and release of a cysteine protease.