Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds.

Abstract

Larvae of the greenbottle fly Lucilia sericata are used routinely for the clinical treatment of difficult necrotic and infected wounds. Degradation by proteinases contained in larval excretory/secretory (ES) products is thought to contribute to wound debridement by removal of dead tissue. However, proteinase activity may also affect host tissue remodelling processes. To identify proteolytic enzymes derived from L. sericata ES products with activities against fibrin and extracellular matrix (ECM) components. Larval proteinase activities were assayed in vitro using class-specific substrates and inhibitors. Their action against fibrin and ECM components was examined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three classes of proteolytic enzyme were detected in the secretions using fluorescein isothiocyanate-labelled casein as a model substrate. The predominant activity belonged to serine proteinases (pH optima 8-9) of two different subclasses (trypsin-like and chymotrypsin-like), with a weaker aspartyl proteinase (pH 5) and a metalloproteinase (pH 9) with exopeptidase characteristics also present. Using skin-relevant ECM components as substrates L. sericata ES products solubilized fibrin clots and degraded fibronectin, laminin and acid-solubilized collagen types I and III. Hydrolysis of ECM macromolecules was inhibited by preincubating ES products with phenylmethylsulphonyl fluoride but not 4-amidinophenylmethylsulphonyl fluoride, indicating that degradation was due to the 'chymotrypsin-like' serine proteinase. These data suggest that a combination of L. sericata ES proteinases involving chymotrypsin-like and trypsin-like activities could potentially influence wound healing events when maggots are introduced into necrotic and infected wounds, with the chymotrypsin-like activity involved in the remodelling of ECM components.