Specific induction of a cuticle-degrading protease of the insect pathogenic fungus Metarhizium anisopliae

Abstract

~The insect pathogenic fungus Metarhizium anisopliae produces several extracellular cuticle-degrading proteases and evidence is consistent that one of these, a chymoelastase PRl, is a determinant of pathogenicity. We have shown previously that the wide-domain regulatory circuits of carbon and nitrogen derepression regulate PRI production. In the present work we have established in addition that PRI is specifically induced by insect cuticle, but not by other soluble or insoluble proteinaceous substrates. The feeding of elastin or collagen to derepressed established mycelium (starved for carbon and nitrogen) did not enhance PR1 production significantly and the soluble proteins BSA and gelatin rapidly and completely repressed PRI. The carbohydrate polymers cellulose and xylan gave derepressed basal levels of PR1. However, addition of locust cuticle enhanced PRI production to a level approximately 10-fold that of derepressed mycelium. In order to establish if the enhancing effect of insect cuticle on PRI production was due to specific induction or merely a reflection of enhanced growth on this insoluble dual carbon and nitrogen source, ergosterol was used as a measure of fungal growth. Expressing enzyme activity per mg dry weight showed that PRI production in cuticle cultures increased approximately five-and ninefold after 12 and 24 h growth compared with elastin-grown cultures. Thus, the substantial increase in PR1 production on cuticle was shown not to be a function of fungal growth and this confirms that PRI is induced by a component of insect cuticle; we believe this is the first report of induction by a specific substrate for any microbial protease.

Atividade proteolítica de isolados de Metarhizium anisopliae sobre substratos cuticulares e não-cuticulares

“…Os resultados obtidos sugerem que, sob as condições testadas, as cutículas de M. fimbriolata e D. flavopicta não demonstraram ser substrato adequado para a detecção de atividade proteolítica em placa (Figura 1). Proteases extracelulares (Pr1 e Pr2) têm sido produzidas por isolados de M. anisopliae quando cultivados em meios líquidos contendo cutículas de diversos insetos, como as dos gafanhotos Schistocerca pallens, Rhammatocerus schistocercoides e Schistocerca gregaria e as da mariposa Manduca sexta (TIAGO et al, 2002;PINTO et al, 2002;PATERSON et al, 1994e ST. LEGER et al, 1988.…”

Genome Organization inBeauveria bassiana:Electrophoretic Karyotype, Gene Mapping, and Telomeric Fingerprint

“…In this work, four nuclear genes amplified by PCR using known conserved primers were cloned. The genes encoded the b-tubulin, the histone 4, the 28S rDNA, and PR1, a protease involved in pathogenicity (Paterson et al, 1994). The PR1 amplification product was sequenced and identified as the PR1 gene.…”

Disruption of the Subtilase Gene, albin1 , in Ophiostoma piliferum

“…The inferred amino acid sequence contains active-site motifs common to protease K subfamily subtilases (42) and is in the size range previously reported for fungal subtilases (20,27). Albin1 groups phylogenetically with extracellular subtilases T, K, R, and Pr1 from M. anisopliae that degrade the cuticle of the exoskeleton of host insects (35). If Albin1 is an extracellular subtilase responsible for the degradation of exogenous proteins, then the albin1 gene may be important for the growth of O. piliferum on wood.…”