Structure-function analysis of PGRP-S1 from the oriental armyworm, Mythimna separata
Structure-function analysis of PGRP-S1 from the oriental armyworm, Mythimna separata
复制标题
东方粘虫 PGRP-S1 的结构-功能分析
DOI：
10.1002/arch.21763
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发表时间：
2021-01-11
影响因子：
2.2
通讯作者：
Rao, Xiang-Jun
中科院分区：
文献类型：
--
作者：
Liu, Fang-Fang;Li, Hao;Rao, Xiang-Jun
肽聚糖识别蛋白（PGRP）是一类能够识别或水解细菌细胞壁主要成分之一肽聚糖（PGN）的蛋白质。然而，对它们的抗真菌活性知之甚少。PGRP-S1以前从作物害虫粘虫（Mythimna separata（步行者））（鳞翅目：夜蛾科）中鉴定。PGRP-S1对革兰氏阳性菌和革兰氏阴性菌均表现出溶菌活性。组织表达分析表明，PGRP-S1主要在幼鱼中肠表达。诱导分析表明，球孢白僵菌分生孢子注射后12 h，在幼虫中肠中有明显的诱导作用。为了确定与其杀菌活性相关的关键残基，预测了PGPR-S1的结构，进行了结构比较和分子对接分析。通过定点诱变将六个残基（H61、H62、Y 97、H171、T175和C179）分别突变为Ala。表达并纯化重组野生型（WT）和突变体蛋白。重组蛋白质结合不同的多糖，PGN和细菌。H61 A、Y 97 A、H171 A和C179 A失去酰胺酶活性。因此，抗菌测定和扫描电子显微镜证实，只有H62 A和T175 A保留溶菌活性。B的萌发。WT、H61 A、Y 97 A、T175 A和C179 A突变体对球孢霉分生孢子的生长有显著的抑制作用。电镜观察发现，处理后部分分生孢子破裂。WT、H61 A、H62 A和T175 A对菌丝生长有抑制作用。综上所述，我们的数据表明PGRP-S1的不同残基参与抗菌和抗真菌活性。
Peptidoglycan recognition proteins (PGRPs) are well known for their abilities to recognize or hydrolyze peptidoglycan (PGN), one of the major bacterial cell wall components. However, much less is known about their antifungal activities. PGRP-S1 was previously identified from a crop pest, Mythimna separata (Walker) (Lepidoptera: Noctuidae). PGRP-S1 showed bacteriolytic activities against Gram-positive and Gram-negative bacteria. In this study, tissue expression analysis showed that PGRP-S1 was mainly expressed in the midgut of naive larvae. The induction analysis showed that it was significantly induced in the larval midgut 12 h post the injection of Beauveria bassiana conidia. To identify the key residues that are related to its microbicidal activities, the structure of PGPR-S1 was predicted for structural comparison and molecular docking analysis. Six residues (H61, H62, Y97, H171, T175, and C179) were mutated to Ala individually by site-directed mutagenesis. The recombinant wild-type (WT) and mutant proteins were expressed and purified. The recombinant proteins bound to different polysaccharides, PGNs, and bacteria. H61A, Y97A, H171A, and C179A lost amidase activity. Accordingly, antibacterial assay and scanning electron microscopy confirmed that only H62A and T175A retained bacteriolytic activities. The germination of B. bassiana conidia was significantly inhibited by WT, H61A, Y97A, T175A, and C179A mutants. Electron microscopy showed that some conidia became ruptured after treatment. The growth of hyphae was inhibited by the WT, H61A, H62A, and T175A. In summary, our data showed that different residues of PGRP-S1 are involved in the antibacterial and antifungal activities.