Weber Brent S, Hennon Seth W, Wright Meredith S, Scott Nichollas E, de Berardinis Véronique, Foster Leonard J, Ayala Juan A, Adams Mark D, Feldman Mario F
Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.
Department of Molecular Microbiology, Washington University School of Medicine in St. Louis, St. Louis, Missouri, USA.
mBio. 2016 Oct 11;7(5):e01253-16. doi: 10.1128/mBio.01253-16.
The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium.
Acinetobacter baumannii is one of the most troublesome and least investigated multidrug-resistant bacterial pathogens. We have previously shown that A. baumannii employs a T6SS to eliminate competing bacteria. Here we provide a comprehensive analysis of the components of the T6SS of Acinetobacter, and our results provide genetic and functional insights into the Acinetobacter T6SS. Through this analysis, we identified a novel peptidoglycan hydrolase, TagX, that is required for biogenesis of the T6SS apparatus. This is the first peptidoglycanase specialized in T6SS function identified in any species. We propose that this enzyme is required for the spatially and temporally regulated digestion of peptidoglycan to allow assembly of the T6SS machinery.
VI型分泌系统（T6SS）是革兰氏阴性菌产生的一种广泛存在的分泌装置，已成为细菌间相互作用中抗菌活性的有效介质。大多数不动杆菌属细菌产生一种基因保守的T6SS，尽管该系统在不同菌株中的表达和功能有所不同。一些致病性鲍曼不动杆菌菌株通过携带T6SS阻遏物的质粒自发丢失来激活该分泌系统。在这项研究中，我们通过转录组测序和差异蛋白质组学比较了有质粒和无质粒细胞中T6SS相关基因的表达。这种方法，结合T6SS簇的突变分析，确定了在医院病原体鲍曼不动杆菌和非病原体拜氏不动杆菌中构建功能性T6SS所需的遗传成分。通过构建T6SS相关vgrG基因发生变化的突变体的全面组合，我们描述了它们对T6SS功能的相对贡献。我们进一步确定了两种效应蛋白（包括一对效应蛋白-免疫蛋白）对抗菌活性的重要性。我们的遗传分析导致鉴定出一种名为TagX的必需膜相关结构成分，我们将其表征为具有l，d-内肽酶活性的肽聚糖水解酶。TagX与已知的噬菌体l，d-内肽酶具有同源性，并且在几种细菌物种的T6SS簇中保守。我们提出TagX是第一个被鉴定出的酶，它在使T6SS机器穿过产生T6SS的细菌的肽聚糖层中发挥重要作用。
鲍曼不动杆菌是最棘手且研究最少的多重耐药细菌病原体之一。我们之前已经表明鲍曼不动杆菌利用T6SS来消除竞争细菌。在这里，我们对不动杆菌T6SS的组成成分进行了全面分析，我们的结果为不动杆菌T6SS提供了遗传和功能方面的见解。通过这项分析，我们鉴定出一种新型肽聚糖水解酶TagX，它是T6SS装置生物合成所必需的。这是在任何物种中鉴定出的第一种专门用于T6SS功能的肽聚糖酶。我们提出这种酶对于肽聚糖的时空调节消化是必需的，以允许T6SS机器的组装。
