Identification and characterisation of the excreted/secreted serine proteases of larvae of the Old World Screwworm Fly, Chrysomya bezziana

Serine proteases are the major proteolytic activity excreted or secreted fr om Chrysoma bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Al a-Pro-Phe-p-nitroanilide. Serine proteases were identified through their in hibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as t rypsin-and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine c hloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectiv ely. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secret ed serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Aminoterminal sequ encing identified both trypsin and chymotrypsin-like sequences in the excre ted or secreted pool with the latter being the dominant protease, whereas t rypsin was the dominant species in the peritrophic matrix eluant. These res ults suggest that trypsin was possibly preferably adsorbed by the peritroph ic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Imm unoblot analysis on dissected gut tissues indicated that the anterior and p osterior midguts were the main source of the serine proteases, although a n ovel species of 32 kDa was predominantly associated with the peritrophic ma trix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological d efence against the parasite. (C) 2000 Australian Society for Parasitology I nc. Published by Elsevier Science Ltd. All rights reserved.