R.J. St. Leger

Cuticle-degrading enzymes of entomopathogenic fungi: Synthesis in culture on cuticle

Abstract Several pathogenic isolates of Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii when grown in buffered liquid cultures containing comminuted locust cuticle as composite carbon source (good growth occurred on most monomeric and polymeric cuticular constituents), produced a variety of extracellular enzymes corresponding to the major components of insect cuticle, e.g., endoproteases, aminopeptidase, carboxypeptidase A, lipase, esterase, chitinase, and N-acetylglucosaminidase. Considerable variations occurred in levels of production between species and even within a species, but endoproteases were exceptional as production of them was high with all the isolates. Cuticle-degrading enzymes were produced rapidly and sequentially in culture. The first activities to appear (

Characterization of cuticle-degrading proteases produced by the entomopathogen Metarhizium anisopliae

Two chymoelastases and three trypsinlike proteases were separated from culture filtrates of the entomopathogen Metarhizium anisopliae. A chymoelastase (Pr1) (pI 10.3 Mr 25,000) and trypsin (Pr2) (pI 4.42, Mr 28,500) were purified to homogeneity by ammonium sulphate precipitation, isoelectric focusing, and affinity chromatography. Inhibition studies showed that both enzymes possessed essential serine and histidine residues in the active site. Pr1 shows greater activity than Pr2 or mammalian enzymes against locust cuticle and also possesses activity vs elastin. Pr1 shows a broad primary specificity toward amino acids with hydrophobic side groups in synthetic ester and amide substrates. The kinetic properties of Pr1 demonstrate a preference for extended peptide chains with the active site recognising at least five substrate residues. The S5 and S4 subsites show a preference for negatively charged succinyl and hydrophobic acetyl groups, respectively. The S3 and S2 subsites both discriminated in favor of alanine and against proline. Pr2 rapidly hydrolyzed casein and synthetic substrates containing arginine or lysine. It possessed little or no activity vs cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. Specific active site inhibitors confirmed the similarities between Pr2 and trypsin.

Production of cuticle-degrading enzymes by the entomopathogen Metarhizium anisopliae during infection of cuticles from Calliphora vomitoria and Manduca sexta

SUMMARY: A biochemical and histochemical investigation with specific substrates and inhibitors was used to visualize protease, esterase and aminopeptidase activities produced in situ during penetration of Calliphora vomitoria and Manduca sexta cuticles by hyphae of the entomopathogenic fungus Metarhizium anisopliae. Two endoproteases, and aminopeptidase and esterase activities, were mainly localized in simple and complex appressoria and germinating conidia. The effect of inhibitors on two characterized proteases (Pr1 and Pr2) and aminopeptidase activity in appressorial plates was quantified by microdensitometric measurement of reaction products. Pr1 and Pr2 activities were differentially inhibited by various protease inhibitors. Pr1, Pr2, esterase, aminopeptidase and N-acetylglucosaminidase (exochitinase) activities were present during penetration as detected directly following desorption from fungal and cuticle components. The proteases produced in situ were fractionated, and were shown by immunological and enzymological criteria to be the same as those produced in culture media. The sequence of enzyme appearance in situ showed that production of proteolytic enzymes precedes exochitinase production. No production of endochitinase was found before or during hyphal penetration of the cuticle.

Cuticle-degrading enzymes of entomopathogenic fungi: Cuticle degradation in vitro by enzymes from entomopathogens

Extracellular fluids from Metarhizium anisopliae, Beauveria bassiana, and Verticillium lecanii grown on cuticle as the sole carbon source released amino acids and N-acetylglucosamine from protein and chitin, respectively, in comminuted locust cuticle. An endoprotease, chitinase, and N-acetyl-β-glucosaminidase were each purified from culture filtrates of M. anisopliae until free of other cuticle-degrading enzymes and tested singly, in combination, or in sequence against “whole cuticle” (containing tanned and untanned proteins) and exuviae (tanned cuticle). The protease hydrolyzed ca. 25–30% of cuticle proteins (ww), releasing peptides (mean chain length, 4.7) containing all 15 amino acids found in locust cuticle. Small amounts of amino sugars were also liberated following protein solubilization. Chitinase tested separately released monomeric N-acetylglucosamine (equivalent to 3–4% of cuticle chitin); however, when combined simultaneously with protease, N-acetylglucosamine release was increased × 1.5. Pretreatment with protease considerably enhanced chitinase activity (ca. × 3.5) compared to controls (preincubated with autoclayed protease). This implies that cuticular chitin is shielded by protein. N-acetyl-β-glucosaminidase showed no detectable activity against cuticle either alone or in combination with protease or chitinase. Exuvia was comparatively resistant to both proteolytic and chitinolytic attack; pretreatment with protease had no effect on subsequent chitinase activity. The results are discussed in relation to cuticle structure and the role of host and fungal enzymes in degrading cuticle during molting or infection.

Role of extracellular chymoelastase in the virulence of Metarhizium anisopliae for Manduca sexta

The relationship between extracellular chymoelastase (Pr1) and the virulence of Metarhizium anisopliae for Manduca sexta was studied using either IgG antibodies vs Pr1 or turkey egg white inhibitor, a potent specific inhibitor of Pr1. Treatment of M. sexta with the inhibitor during infection significantly delayed mortality. The inhibitor also reduced melanization of cuticle (an early host response to infection) and invasion of the hemolymph, as well as maintained the hosts growth rate. The incorporation of antibodies or inhibitor delayed penetration of cuticle, but did not affect spore viability or prevent growth and formation of appressoria on the cuticle surface. This suggests that inhibition of Pr1 reduced infection by limiting fungal penetration of the insect cuticle. In vitro studies using inhibitor showed that the accumulation of protein degradation products from cuticle, including ammonia, was dependent on active Pr1. This confirms the major role of Pr1 in solubilizing cuticle proteins and making them available for nutrition. It is concluded that Pr1 serves as a virulence factor by causing the localized destruction of cuticle proteins, which enables rapid invasion of the host with concomitant provision of nutrients.

Cuticle-degrading enzymes of entomopathogenic fungi: Mechanisms of interaction between pathogen enzymes and insect cuticle

Abstract Extracellular enzymes of Metarhizium anisopliae had considerable affinity for insect cuticle. Binding of proteases immobilized over 70% of soluble enzyme activity, which in vivo could have a significant influence on the extent and nature of cuticle degradation. Adsorbed protease, carboxypeptidase, and N-acetylglucosaminidase activities were recoverable with 0.2 m buffer suggesting nonspecific ionic binding. Chitinase bound irreversibly as a specific enzyme-substrate complex. Cuticle degradation by an alkaline (optimum pH 9) basic (pI 9.5) protease was inhibited by increasing salt concentrations while anilide hydrolysis was unaffected. Inhibition arose from interference with essential electrostatic adsorption of the enzyme on to the cuticle. An anionic detergent enhanced enzymic solubilization of cuticle proteins (probably due to increased electronegativity of cuticle) at the expense of continued proteolysis of released peptides, clearly distinguishing between the two processes. A cationic detergent inhibited cuticle degradation, indicating that salt labile bonds form between the negative (probably carboxyl) groups of cuticle and the positively charged groups of the protease. The significance of these results in understanding the mechanism of cuticle degradation are discussed.

Distribution of chymoelastases and trypsin-like enzymes in five species of entomopathogenic deuteromycetes

Nine isolates of the entomopathogenic deuteromycetes Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Nomuraea rileyi, and Aschersonia aleyrodis produced basic (pI greater than 7.0) chymoelastases that possessed extended binding sites, comprising at least four or five subsites, with preference for hydrophobic residues at the primary binding site. Most isolates also produced additional acidic enzymes with similar specificities against ester and amide substrates but which lacked activity against elastin. Both acidic and basic enzymes degraded high protein azure or locust cuticle and, as shown by inhibition studies, possessed essential serine and histidine residues in the active site. In spite of similarities in catalytic properties antibodies generated against a Metarhizium chymoelastase cross-reacted only with enzymes from two (out of four) Metarhizium isolates; enzymes from all other isolates did not cross-react. Two isolates of Metarhizium produced a third class of protease which degraded Bz-AA-AA-Arg-NA substrates (AA, various amino acids) and hide protein azure. Analogous peptidases were produced by other isolates but they were specific for Bz-Phe-Val-Arg-NA and showed less sensitivity to trypsin inhibitors. The possible significance to pathology of the presence of diverse yet similar protease forms in five genera of entomopathogens is discussed.

Regulation of production of proteolytic enzymes by the entomopathogenic fungus Metarhizium anisopliae

Synthesis of chymoelastase and trypsin by the entomopathogenic fungus Metarhizium anisopliae occurs rapidly (<2 h) during carbon and nitrogen derepression in minimal media. Enzyme levels were enhanced when minimal media were supplemented with insect cuticle or other insoluble polymetic nutrients (e.g. cellulose) that were insufficient to produce catabolite repression. Addition of more readily utilized metabolites (e.g. glucose or alanine) repressed protease production confirming that production is constitutive but repressible. Operational control of protease release involves synthesis rather than secretion because catabolite repression reduced endocellular activity (associated with a sedimentable vacuole containing fraction) as well as extracellular enzyme levels. Studies with metabolic inhibitors indicated that production of Pr1 and Pr2 does not require DNA synthesis. However, synthesis is substantially reduced by inhibitors of transcription (actinomycin D and 8-azoguanine) and translation (cyclohexamide and puromycin).Inhibition by 8-azoguanine is relieved by guanine. These results imply that the operative steps in protease regulation involve de novo synthesis of mRNA. Inhibition of enzyme production by an AMP analogue adenosine 5′-0-thiophosphate implies an involvement for AMP-dependent enzyme systems in derepression. However, neither exogenous cAMP nor an inhibitor of cAMP phosphodiesterase relieved catabolite repression by glucose or NH4Cl. Use of o-vanadate to inhibit plasmalemma ATPase confirmed that secretion of chymoelastase-like protease and trypsin-like protease via the cell membrane is an active process.

Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae

Abstract Extracellular fluids from Metarhizium anisopliae grown on chitin as the sole carbon source contained distinct chitinase ( p H optimum 5.3, MW 33 kDa) and chitobiase ( p H optimum 5, MW 110 kDa, p I 6.4) activities. Chitinase activity was stabilized against extremes of p H and temperature by the presence of its chitin substrate. Chitinase fractions eluted from a sephadex column had activity against chitosan as well as locust cuticle chitin, crystalline chitin, and colloidal chitin. Products of enzymolysis were analyzed by descending paper chromatography. Chitinase had no activity against chitobiose, only trace activity against chitotriose, but preferentially cleaved chitotetraose in the middle bond releasing chitobiose. N -Acetyglucosamine was the major (vs colloidal chitin) or only (vs crystalline chitin) product detected following chitin hydrolysis. The chitobiase (also called β- N -acetylglucosaminidase) possessed simple glycosidase activity against p -nitrophenyl-acetylglucosaminide and hydrolyzed di-, tri-, and tetrasaccharides to N -acetylglucosamine. Chitobiase activity was significantly inhibited by its reaction product, N -acetylglucosamine. The mechanism of action of chitinolytic enzymes is discussed.

The effect of melanization of Manduca sexta cuticle on growth and infection by Metarhizium anisopliae

Growth and chymoelastase (Pr1) production by Metarhizium anisopliae were far greater in shake cultures containing unpigmented cuticle than on melanin-cuticle complexes (prepared from reacting l-DOPA or catechol with Manduca sexta cuticle containing tyrosinase). Poor growth with the melanin-cuticle complexes mainly resulted from toxic effects from catechol and l-DOPA oxidation products including melanin; also, melanin may partially shield cuticle from enzymatic attack. Such protection is incomplete as pure Pr1 releases melanin from cuticle by hydrolysis of the associated protein. Pr1 activity also contributed to solubilization of cuticle-bound melanin during growth; addition of a specific Pr1 inhibitor reduced solubilized melanin after 6 days by 43%. Pr1 and Pr2 (M. anisopliae trypsin) did not interact with M. sexta tyrosinase, nor (in contrast to bovine chymotrypsin) were they inhibited by incubation in a melanizing mixture of tyrosinase and l-DOPA. H2O2 and ·OH which are potentially active against cuticle polymers including melanin were not detected in culture filtrates which, however, contained a H2O2-splitting activity (catalase). In contrast, extracellular H2O2 was formed by isolates of M. anisopliae, Beauveria bassiana, and Verticillium lecanii on a solid detection medium.