Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Abstract

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.

The PacC transcription factor regulates secondary metabolite production and stress response, but has only minor effects on virulence in the insect pathogenic fungus Beauveria bassiana

“…Indeed, the fungal-insect cuticle surface interaction has been theorized as being the critical interface leading to successful mycosis and one on which an evolutionary arms race between an insect and B. bassiana has been described (Ortiz-Urquiza and Keyhani, 2013; Pedrini et al, 2015). During infection by entomopathogenic fungi, an increase in cuticular pH occurs, in part due to the production of ammonia by the fungus (St Leger et al, 1998;Jin et al, 2010). Ammonia has recently been shown to activate pacC in the plant pathogen Penicillium expansum during colonization of apple .…”

“…citrate, malate, succinate, formate, and acetate). In M. anisopliae, acid-non-producing mutants showed limited growth under alkaline conditions, indicating that acidification of the media is linked to the ability of the fungus to grow at elevated pH (St Leger, et al, 1998). Thus, alkalinization of the host cuticle during the initial phases of infection may activate PacC leading to the production of oxalate (acidification of the media) and other factors required for further growth, penetration and subsequent ingress into the host.…”

“…Molecular characterization of pathways important for B. bassiana virulence has shown that both transcription and the enzymatic activities of many of these enzymes, e.g. chitinases, proteases, and lipases, as well as other fungal systems, are modulated by environmental pH, (St Leger et al, 1998;. In turn, entomopathogenic fungi are capable of modulating their external pH microenvironment in order to induce the expression of specific secreted enzymes, e.g.…”

PacC in the nematophagous fungus Clonostachys rosea controls virulence to nematodes

“…The importance of pH regulation in the expression of genes encoding putative virulence factors has been shown in a variety of plant, insect and mammalian pathogens. These virulence factors, which are affected by ambient pH, include cell wall-degrading enzymes, such as chitinase, N-acetylglucosaminidase (St Leger et al, 1998), polygalacturonases (Wubben et al, 2000;Caracuel et al, 2003), endoglucanase (Eshel et al, 2002), secreted enzymes such as proteases (St Leger et al, 1998) and pectate lyase (Yakoby et al, 2000). Previously, we have purified a subtilisin-like extracellular protease PrC and cloned its gene from the fungus C. rosea (Li et al, 2006) Zhang, unpubl.…”

“…It has been shown that PacC/Rim101 controls fungal pathogenicity to plants in Fusarium oxysporum (Caracuel et al, 2003), Sclerotinia sclerotiorum (Rollins, 2003) and Colletotrichum acutatum (You et al, 2007), and to mammals by Candida albicans (Davis, 2003) and Aspergillus nidulans (Bignell et al, 2005). Although a previous study has reported that subtilisin-like extracellular proteases in the insect pathogen M. anisopliae are expressed under alkaline growth conditions (St Leger et al, 1998), the role of PacC in the regulation of these genes has not been established. In this paper, we isolated a pacC orthologue in C. rosea and investigated the role of PacC-mediated pH signalling in virulence.…”