DEVELOPMENTALLY-REGULATED SECRETION OF CATHEPSIN L-LIKE CYSTEINE PROTEASES BY HAEMONCHUS-CONTORTUS

Cysteine protease activity was present in media collected after 24 hr in vitro culture of adult Haemonchus contortus. The released cysteine protease hydrolyzed the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-substituted synthetic peptides Z-phe-arg-AFC and Z-ala-arg-arg-AFC, but not Z-arg-arg-AFC or Z-arg-AFC, characterizing this activity as cathepsin L-like. Within the parasite, cysteine protease activity was highest in extracts of intestinal tissue. Secreted cysteine protease inhibited the clotting of sheep blood and hydrolyzed hemoglobin, fibrinogen, collagen, and IgG; the IgG hydrolysis site was within the hinge region. Four proteases with M(r) values of 30, 34, 37, and 41 kDa were identified with biotinylated-phenylalanine-arginine-fluoromethyl ketone, a specific probe that binds to active cysteine proteases. Adult parasites cultivated in the presence of 0.1 mM levamisole released 50% less protease activity compared to control cultures; in the presence of rafoxanide (0.1 mM), protease was not detected. Cathepsin L-like cysteine protease activity was released also by L4, but not the L3 larval stage. The active and developmentally regulated release of cysteine proteases by H. contortus may have a critical function in worm nutrition, immune evasion, or both.