Secretome analysis of Vibrio cholerae type VI secretion system reveals a new effector-immunity pair.
Secretome analysis of Vibrio cholerae type VI secretion system reveals a new effector-immunity pair.
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DOI：
10.1128/mbio.00075-15
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发表时间：
2015-03-10
期刊：
影响因子：
6.4
通讯作者：
Mekalanos J
中科院分区：
文献类型：
--
作者：
Altindis E;Dong T;Catalano C;Mekalanos J
VI型分泌系统（T6 SS）是一种动态的大分子细胞器，许多革兰氏阴性细菌使用它来抑制或杀死其他原核或真核细胞。T6 SS的毒性效应物以接触依赖性方式递送到猎物细胞。在霍乱弧菌（霍乱的病原体）中，T6 SS在肠道感染期间是活跃的。在这里，我们描述了使用比较蛋白质组学与生物信息学相结合来识别新的T6 SS效应-免疫对。该分析能够鉴定除含有帕尔（脯氨酸、丙氨酸、丙氨酸、精氨酸）基序的蛋白以外的所有先前鉴定的T6 SS分泌底物。此外，该方法导致鉴定了由VCA 0285（TseH）编码的携带预测的水解酶结构域的新分泌蛋白。我们证实了TseH在大肠杆菌和霍乱弧菌细胞的周质中表达时是有毒的。在霍乱弧菌中观察到的毒性被VCA 0286（TsiH）编码的蛋白质的共表达抑制，表明该蛋白质是TseH的同源免疫蛋白。此外，向透化的大肠杆菌细胞中外源添加纯化的重组TseH引起细胞裂解。TseH蛋白序列的生物信息学分析表明，它是一个新的家庭的细胞壁降解酶，包括属于YD重复和Rhs超家族的蛋白质的成员，TseH的直系同源物可能表达属于门不同的拟杆菌和变形菌。革兰氏阴性细菌霍乱弧菌引起霍乱，这是一种严重且往往致命的霍乱疾病。2010-2012年在海地发生的疫情和非洲新爆发的疫情表明，霍乱仍然是一个重大的全球公共卫生问题。VI型分泌系统（T6 SS）是由许多革兰氏阴性菌表达的动态细胞器，其用于将毒性效应蛋白注入真核细胞和细菌猎物细胞。在这项研究中，我们应用比较蛋白质组学的方法，以确定新的基板，这种分泌装置的霍乱弧菌T6 SS分泌组。我们表明，VCA 0285基因的产物可能是一种新的肽聚糖水解酶，由T6 SS分泌，其同源免疫蛋白由直接下游的基因（VCA 0286）编码。生物信息学分析表明，VCA 0285携带四个保守的基序，可能定义了一个大家族的水解酶具有抗菌活性。新的抗菌T6 SS效应物的鉴定为开发新的抗生素和治疗剂提供了有用的信息。
The type VI secretion system (T6SS) is a dynamic macromolecular organelle that many Gram-negative bacteria use to inhibit or kill other prokaryotic or eukaryotic cells. The toxic effectors of T6SS are delivered to the prey cells in a contact-dependent manner. In Vibrio cholerae, the etiologic agent of cholera, T6SS is active during intestinal infection. Here, we describe the use of comparative proteomics coupled with bioinformatics to identify a new T6SS effector-immunity pair. This analysis was able to identify all previously identified secreted substrates of T6SS except PAAR (proline, alanine, alanine, arginine) motif-containing proteins. Additionally, this approach led to the identification of a new secreted protein encoded by VCA0285 (TseH) that carries a predicted hydrolase domain. We confirmed that TseH is toxic when expressed in the periplasm of Escherichia coli and V. cholerae cells. The toxicity observed in V. cholerae was suppressed by coexpression of the protein encoded by VCA0286 (TsiH), indicating that this protein is the cognate immunity protein of TseH. Furthermore, exogenous addition of purified recombinant TseH to permeabilized E. coli cells caused cell lysis. Bioinformatics analysis of the TseH protein sequence suggest that it is a member of a new family of cell wall-degrading enzymes that include proteins belonging to the YD repeat and Rhs superfamilies and that orthologs of TseH are likely expressed by species belonging to phyla as diverse as Bacteroidetes and Proteobacteria. The Gram-negative bacterium Vibrio cholerae causes cholera, a severe and often lethal diarrheal disease. The 2010-2012 epidemic in Haiti and new explosive epidemics in Africa show that cholera remains a significant global public health problem. The type VI secretion system (T6SS) is a dynamic organelle expressed by many Gram-negative bacteria, which use it to inject toxic effector proteins into eukaryotic and bacterial prey cells. In this study, we applied a comparative proteomics approach to the V. cholerae T6SS secretome to identify new substrates of this secretion apparatus. We show that the product of the gene VCA0285 is likely a new peptidoglycan hydrolase that is secreted by T6SS and that its cognate immunity protein is encoded by the gene that is immediately downstream (VCA0286). Bioinformatics analysis shows that VCA0285 carries four conserved motifs that likely define a large family of hydrolases with antibacterial activity. The identification of new antibacterial T6SS effectors provides useful information for the development of novel antibiotics and therapeutic agents.