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

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.

分泌蛋白水解酶与几丁质水解酶，是金龟子绿僵菌（Metarhizium anisopliae）响应宿主（昆虫）表皮信号启动侵染过程的标志性特征。本研究通过Northern印迹分析（Northern analysis）与/或酶活测定，对上述酶类包括类枯草杆菌蛋白酶（subtilisin-like proteases）Pr1a、Pr1b，类胰蛋白酶（trypsin-like proteases）Pr2，金属蛋白酶（metalloproteases），天冬氨酸蛋白酶（aspartyl proteases），氨肽酶（aminopeptidase）以及几丁质酶（chitinases）以及一种疏水蛋白（hydrophobin）的调控机制展开了研究。各类酶的合成均呈现出响应环境pH的差异化表达模式：仅当培养基pH处于该酶的有效作用范围时，相关酶才会被合成，且这一规律不受培养基中是否添加诱导性表皮底物的影响。三种天冬氨酸蛋白酶（最适pH为3）以及几丁质酶（最适pH为5）在酸性pH条件下的积累量达到峰值。氨肽酶（最适pH为7）的表达量在pH7时达到峰值。五种金属蛋白酶（最适pH约为7）的最高表达量出现在pH6至8的范围内。两种类胰蛋白酶以及数种最适pH约为8的类枯草杆菌蛋白酶Pr1同工型，仅在碱性条件下得以合成。针对7个编码蛋白酶与几丁质酶的互补DNA（cDNA）序列对应的RNA转录本进行的Northern印迹分析证实，环境pH对分泌蛋白的基因表达具有关键调控作用。疏水蛋白在pH5与pH8条件下的表达量基本持平，但在pH3条件下不表达。在真菌侵染宿主的过程中，被侵染表皮的pH值会从约6.3上升至7.7。这与酶分泌的pH调控规律相符：侵染过程中丝氨酸蛋白酶与金属蛋白酶可在原位被检测到，但未发现天冬氨酸蛋白酶的表达。本研究提出，被侵染表皮的碱化可作为一种生理信号，触发毒力因子的产生。