Academy of Agricultural Sciences, Southwest University, Chongqing, People's Republic of China
Biotechnology Research Center, Southwest University, Chongqing, People's Republic of China.
Appl Environ Microbiol. 2018 Jul 17;84(15). doi: 10.1128/AEM.01086-18. Print 2018 Aug 1.
Fungal β-1,3-glucanosyltransferases are cell wall-remodeling enzymes implicated in stress response, cell wall integrity, and virulence, with most fungal genomes containing multiple members. The insect-pathogenic fungus displays robust growth over a wide pH range (pH 4 to 10). A random insertion mutant library screening for increased sensitivity to alkaline (pH 10) growth conditions resulted in the identification and mapping of a mutant to a β-1,3-glucanosyltransferase gene (). expression was pH dependent and regulated by the PacC transcription factor, which activates genes in response to neutral/alkaline growth conditions. Targeted gene knockout of resulted in reduced growth under alkaline conditions, with only minor effects of increased sensitivity to cell wall stress (Congo red and calcofluor white) and no significant effects on fungal sensitivity to oxidative or osmotic stress. The cell walls of Δ aerial conidia were thinner than those of the wild-type and complemented strains in response to alkaline conditions, and β-1,3-glucan antibody and lectin staining revealed alterations in cell surface carbohydrate epitopes. The Δ mutant displayed alterations in cell wall chitin and carbohydrate content in response to alkaline pH. Insect bioassays revealed impaired virulence for the Δ mutant depending upon the pH of the media on which the conidia were grown and harvested. Unexpectedly, a decreased median lethal time to kill (LT, i.e., increased virulence) was seen for the mutant using intrahemocoel injection assays using conidia grown at acidic pH (5.6). These data show that BbGas3 acts as a pH-responsive cell wall-remodeling enzyme involved in resistance to extreme pH (>9). Little is known about adaptations required for growth at high (>9) pH. Here, we show that a specific fungal membrane-remodeling β-1,3-glucanosyltransferase gene () regulated by the pH-responsive PacC transcription factor forms a critical aspect of the ability of the insect-pathogenic fungus to grow at extreme pH. The loss of resulted in a unique decreased ability to grow at high pH, with little to no effects seen with respect to other stress conditions, i.e., cell wall integrity and osmotic and oxidative stress. However, pH-dependent alternations in cell wall properties and virulence were noted for the Δas3 mutant. These data provide a mechanistic insight into the importance of the specific cell wall structure required to stabilize the cell at high pH and link it to the PacC/Pal/Rim pH-sensing and regulatory system.
真菌β-1,3-葡聚糖转移酶是一种细胞壁重塑酶，涉及应激反应、细胞壁完整性和毒力，大多数真菌基因组包含多个成员。昆虫病原真菌 在 pH 值为 4 到 10 的较宽范围内具有强劲的生长能力。通过对碱性（pH10）生长条件下敏感性增加的随机插入突变体文库进行筛选，鉴定并定位了一个突变体到一个β-1,3-葡聚糖转移酶基因（）。表达受 pH 影响，并受 PacC 转录因子调控，该因子激活响应中性/碱性生长条件的基因。靶向基因敲除导致碱性条件下生长减少，对细胞壁应激（刚果红和钙荧光白）的敏感性增加仅略有影响，对真菌对氧化或渗透应激的敏感性没有显著影响。与野生型和互补菌株相比，在碱性条件下，Δ气生分生孢子的细胞壁更薄，β-1,3-葡聚糖抗体和凝集素染色显示细胞表面碳水化合物表位发生改变。Δ突变体在碱性 pH 下改变了细胞壁几丁质和碳水化合物含量。昆虫生物测定显示，根据分生孢子生长和收获的培养基的 pH，Δ突变体的毒力受损。出乎意料的是，在用酸性 pH（5.6）生长的分生孢子进行的血腔注射测定中，突变体的中位致死时间（LT，即毒力增加）降低。这些数据表明，BbGas3 作为一种 pH 响应性细胞壁重塑酶，参与抵抗极端 pH（>9）。对于在高 pH（>9）下生长所需的适应知之甚少。在这里，我们表明，一种由 pH 响应性 PacC 转录因子调控的特定真菌膜重塑β-1,3-葡聚糖转移酶基因（）是昆虫病原真菌在极端 pH 下生长的关键因素。丧失导致在高 pH 下生长的能力独特降低，而对细胞壁完整性和渗透和氧化应激等其他应激条件几乎没有影响。然而，观察到Δas3 突变体的细胞壁特性和毒力发生 pH 依赖性改变。这些数据提供了对特定细胞壁结构的重要性的机制见解，该结构需要在高 pH 下稳定细胞，并将其与 PacC/Pal/Rim pH 感应和调节系统联系起来。
