Production and Regulation of Cuticle-Degrading Proteases from Beauveria bassiana in the Presence of Rhammatocerus schistocercoides Cuticle

Abstract

Extracellular proteases have been shown to be virulence factors in fungal pathogenicity toward insects. We examined the production of extracellular proteases, subtilisin-like activity (Pr1) and trypsin-like activity (Pr2), by Beauveria bassiana CG425, which is a fungus of interest for control of the grasshopper Rhammatocerus schistocercoides. To access the role of these proteases during infection of R. schistocercoides, we analyzed their secretion during fungus growth either in nitrate-medium or in cuticle-containing medium supplemented with different amino acids. The enhancing effect of cuticle on Pr1 and Pr2 production suggests that these protease types may be specifically induced by components of the grasshopper cuticle. In medium supplemented with methionine a high level of Pr1 was observed. The remaining amino acids tested did not induce the protease to the levels seen with cuticle. The amino acid methionine seems to play a regulatory role in Pr1 secretion by B. bassiana, since both induction and repression seem to be dependent on the concentration of the amino acid present in the culture medium.

CYP52X1, Representing New Cytochrome P450 Subfamily, Displays Fatty Acid Hydroxylase Activity and Contributes to Virulence and Growth on Insect Cuticular Substrates in Entomopathogenic Fungus Beauveria bassiana

“…As a broad host range pathogen, strains of B. bassiana have been exploited for use against agricultural pests and for insects that act as human and animal disease vectors (1)(2)(3)(4). Infection of insects begins with attachment of fungal spores to the cuticles of target hosts, and in response to surface cues the fungus germinates; the emerging germ tubes produce a variety of enzymes that combined with mechanical pressure begins the process of cuticle penetration (5)(6)(7)(8)(9). The outermost layer of the insect cuticle, also referred to as the waxy layer, serves as the first barrier against microbial attack and consists of a heterogeneous mixture of lipids that include long-chain alkanes, alkenes, wax esters, and fatty acids (10).…”

Production of cuticle-degrading proteases by Nomuraea rileyi and its virulence against Anticarsia gemmatalis

“…Similarly, in previous studies, N. rileyi demonstrated increased proteolytic activity in the presence of Manduca sexta, Trichoplusia ni and Helicoverpa zea cuticles (ST. LEGER et al, 1987a;EL SAYED et al, 1993a). Similar results were observed for other entomopathogenic fungi (ST. LEGER et al, 1989;GUPTA et al, 1992;PATERSON et al, 1994;GILLESPIE et al, 1998;TIAGO et al, 2002;PINTO et al, 2002;CAMPOS et al, 2005;DONATTI et al, 2008;DIAS et al, 2008). Analyses of the proteolytic and chitinolytic activities of M. anisopliae and B. bassiana, performed in media containing different sources of carbon/ nitrogen and complex substrates such as colloidal chitin and cuticle of different insects showed more activities of these enzymes in the media containing complex substrates, suggesting that the expression of the genes involved in the synthesis of Pr1 and Pr2, as well as that for chitinases, depends on the level of carbon and nitrogen sources in the culture medium (MORAES, et al, 2003;CAMPOS, et al, 2005, TIAGO et al, 2007DONATTI et al, 2008).…”

“…The entomopathogenic fungus B. bassiana showed high PR1 and PR2 enzyme activities when cultured in medium containing Rhammatocerus schistocercoides grasshopper cuticle (DONATTI et al, 2008) and in medium containing Hypothenemus hampei coffee berry borer cuticle (DIAS et al, 2008).…”

Selection, assessment of virulence to Alphitobius diaperinus, and Pr1 enzyme production of Beauveria bassiana (Bals.) Vuill. isolates cultured at stress temperatures

“…The enzymatic reactions were performed following the protocol described by Donatti et al (2008). The protocol used for enzymatic quantification is based on hydrolysis of the substrate and the release of p-nitroaniline, which absorbs at a wavelength of 410 nm.…”