A study on cuticle-degrading enzymes (CDE) of three hyphomycete entomopathogens has produced information on enzyme types, levels, characteristics, mode of action, regulation, sequence of production, cellular localisation and production during host penetration. This is the first critical work on CDE of any entomopathogen. Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as sole carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular and bound enzymes corresponding to the major components of insect cuticle e.g. 3 endo-proteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase and N-acetylglucosaminidase. Considerable variations occurred in levels of production between spp. and even within a sp., but endo-proteases were exceptional in being produced in large amounts by all the isolates. CDE were produced rapidly and sequentially in culture. The first activities to appear (<24 h) were those of the proteolytic complex, chitinases were always produced substantially later.Properties of CDE were investigated in terms of pH and tem- erature optima, substrate specificity, molecular weight, iso-electric point, mechanism of substrate degradation and the effect of specific inhibitors. Studies with culture filtrates and purified CDE revealed that substrates in intact cuticles are amenable to degradation but the prior action of protease is necessary for significant degradation of the chitin. Staining of chitin by a fluorescent lectin (FITC-WGA) and calcofluor only in cuticles from which protein has been removed (by protease or KOH) also suggests initial masking of chitin. This and determination of amino acid composition of peptides solubilised by endo-protease revealed the potential of CDE in studying the physicochemical structure of insect cuticles. The apparently localised action of CDE during host penetration may result from molecular sieving, binding to fungal walls or binding to cuticle. The first possibility is lessened by the small size of the endo-enzymes (<34 K daltons) which could allow diffusion via the various canals which traverse cuticle. However, cuticle effectively binds (ionically) CDE, and also activities of several CDE remain partly bound in various ways to hyphae and conidia (by ionic binding to walls, by disulphide bonds, and on or within membrane structures).The involvement of proteolytic enzymes in infection was suggested by their presence in conidia, penetration structures, and infected cuticle (detected histochemically and following extraction from cuticles). Also the constitutive production of endo- and exo-proteases lends weight to their possible significance in parasitism as synthesis will be subject only to catabolite repression. Chitinase is induced by N-acetylglucosamine and was not detected in infected cuticle. Possible mechanisms and significance of enzymic degradation of cuticle during infection are discussed, particularly in compari-son to host penetration by phytopathogenic fungi.
|Date of Award||1985|