Aly Khaled A, Anderson Mark, Ohr Ryan Jay, Missiakas Dominique
Department of Microbiology, University of Chicago, Chicago, Illinois, USA.
Department of Microbiology, University of Chicago, Chicago, Illinois, USA
J Bacteriol. 2017 Oct 31;199(23). doi: 10.1128/JB.00482-17. Print 2017 Dec 1.
The ESAT6-like secretion system (ESS) of promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for establishment of persistent abscess lesions and the modulation of immune responses during bloodstream infections. However, the biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are unknown. Earlier work demonstrated that deletion of , which encodes a membrane protein, abolishes ESS secretion. Loss-of-function mutations truncating the gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB is a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD, and EsxA. All five proteins are essential for secretion, as knockout mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD. Type VII secretion systems support the lifestyle of Gram-positive bacteria, including important human pathogens such as , , and Genes encoding SpoIIIE-FtsK-like ATPases and WXG100-secreted products are conserved features of type VII secretion pathways; however, most of the genes in T7SS clusters are not conserved between different bacterial species. Here, we isolate a complex of proteins from the membranes of that appears to represent the core secretion machinery, designated ESS. These results suggest that three membrane proteins, EsaA, EssB, and EssA, form a secretion complex that associates with EssC, the SpoIIIE-FtsK-like ATPase, and with EsxA, a mobile machine component and member of the WXG100 protein family.
的ESAT6样分泌系统（ESS）促进效应蛋白穿过细菌包膜的运输。ESS簇中的基因对于持续性脓肿病变的形成以及血流感染期间免疫反应的调节是必需的。然而，大多数ESS基因产物的生化功能，特别是分泌机器组件的身份，尚不清楚。早期的研究表明，编码膜蛋白的基因缺失会消除ESS分泌。截短基因产物的功能丧失突变在ESS分泌上导致显性负性表型，表明EssB是分泌机器的核心组件。为了验证这一预测，我们通过十二烷基麦芽糖苷提取从葡萄球菌膜中纯化了天然的和带有亲和标签的EssB，鉴定出一个由EsaA、EssA、EssB、EssD和EsxA五种蛋白质组装而成的复合物。所有这五种蛋白质对于分泌都是必需的，因为相应基因中的敲除突变会消除ESS运输。生化和细菌双杂交分析揭示了EssB和EsaA之间的直接相互作用，通过与移动机器组件EsxA结合，还可能招募EssA和EssD。VII型分泌系统支持革兰氏阳性菌的生存方式，包括重要的人类病原体，如、和。编码SpoIIIE - FtsK样ATP酶和WXG100分泌产物的基因是VII型分泌途径的保守特征；然而，T7SS簇中的大多数基因在不同细菌物种之间并不保守。在这里，我们从的膜中分离出一种蛋白质复合物，它似乎代表了核心分泌机器，命名为ESS。这些结果表明，三种膜蛋白EsaA、EssB和EssA形成一个分泌复合物，该复合物与SpoIIIE - FtsK样ATP酶EssC以及移动机器组件和WXG100蛋白家族成员EsxA相关联。
