iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus
iTRAQ-based proteomic analysis of LI-F type peptides produced by Paenibacillus polymyxa JSa-9 mode of action against Bacillus cereus
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基于 iTRAQ 的蛋白质组学分析多粘类芽孢杆菌 JSa-9 产生的 LI-F 型肽对蜡状芽孢杆菌的作用模式
DOI：
10.1016/j.jprot.2016.08.019
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发表时间：
2017-01-06
影响因子：
3.3
通讯作者：
Lv, Fengxia
中科院分区：
文献类型：
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作者：
Han, Jinzhi;Gao, Peng;Lv, Fengxia
LI-F型肽（AMP-jsa9）是由多粘类芽孢杆菌JSa-9产生的一类环脂肽抗生素，对革兰氏阳性菌和丝状真菌，特别是蜡样芽孢杆菌和镰刀菌具有广泛的抗菌谱。为了更好地了解AMP-jsa9对蜡样芽孢杆菌的抑菌机制，本研究采用原子力显微镜和透射电镜观察AMP-jsa9处理后的蜡样芽孢杆菌细胞的超微结构，并采用iTRAQ （isobaric tag for relative and absolute quantitation）标记和LC-MS/MS分析对处理后和未处理的芽孢杆菌细胞中提取的蛋白进行定量蛋白质组学分析，获取差异表达蛋白。此外，通过实时荧光定量PCR检测ATP、NAD((+))H、NADP((+))H、活性氧（ROS）、过氧化氢酶（CAT）和超氧化物歧化酶（SOD）活性以及靶基因的相对表达，对蛋白质组学分析结果进行验证。暴露于AMP-jsa9的细菌细胞表面呈不规则状，有泡状突起和凹形；我们假设AMP-jsa9穿透细胞壁并锚定在细胞质膜上，并且经过AMP-jsa9处理后，ROS在细胞膜上积累，调节了细菌的膜特性，增加了膜的通透性。因此，在细胞间内容物渗漏的冲力作用下，在细胞壁上形成气泡。基于itraq的蛋白质组学分析共检测到1317个蛋白，其中176个差异表达蛋白（75个上调（fold bbbb2））和101个下调（fold bbbb2）
LI-F type peptides (AMP-jsa9) produced by Paenibacillus polymyxa JSa-9 are a group of cyclic lipodepsipeptide antibiotics that exhibit a broad antimicrobial spectrum against Gram-positive bacteria and filamentous fungi, especially Bacillus cereus and Fusarium moniliforme. In this study, to better understand the antibacterial mechanism of AMP-jsa9 against B. cereus, the ultrastructure of AMP-jsa9-treated B. cereus cells was observed by both atomic force microscopy and transmission electron microscopy, and quantitative proteomic analysis was performed on proteins extracted from treated and untreated bacterial cells by using isobaric tag for relative and absolute quantitation (iTRAQ) labeling and LC-MS/MS analysis to access differentially expressed proteins. Furthermore, multiple experiments were conducted to validate the results of the proteomic analysis, including determinations of ATP, NAD((+))H, NADP((+))H, reactive oxygen species (ROS), the activities of catalase (CAT) and superoxide dismutase (SOD), and the relative expression of target genes by quantitative real-time PCR. Bacterial cells exposed to AMP-jsa9 showed irregular surfaces with bleb projections and concaves; we hypothesize that AMP-jsa9 penetrated the cell wall and was anchored on the cytoplasmic membrane and that ROS accumulated in the cell membrane after treatment with AMP-jsa9, modulating the bacterial membrane properties and increasing membrane permeability. Consequently, the blebs were formed on the cell wall by the impulsive force of the leakage of intercellular contents. iTRAQ-based proteomic analysis detected a total of 1317 proteins, including 176 differentially expressed proteins (75 upregulated (fold >2) and 101 downregulated (fold