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Mechanism of type VI protein secretion

VI型蛋白分泌机制

基本信息

批准号：
194363196
负责人：
Privatdozent Dr. Axel Mogk
金额：
--
依托单位：
Zentrum für Molekulare Biologie (ZMBH)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2011
资助国家：
德国
起止时间：
2010-12-31 至 2013-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/194363196?language=en
关键词：
Mechanism type VI protein secretion

项目摘要

Type VI secretion systems (T6SSs) are found in many pathogenic Gram-negative bacteria and contribute to virulence in various species by delivering effectors into target cells. Exoproteins of T6SSs (Hcp, VgrG) exhibit structural similarities to components of the cell puncturing device of tailed bacteriophages. It is therefore suggested that T6SSs represent contractile injection machineries, thereby using a unique mechanism to target toxins into host cells. We could demonstrate that the AAA+ chaperone ClpV acts as an essential ATPase of T6SSs by severing VipA/VipB tubules, which are conserved and essential components of T6SSs. The architecture of VipA/VipB tubules suggests that they might function in analogy to tail sheath proteins of bacteriophages, driving the ejection of the T6SS exoproteins through contraction. We propose that such conformational change of VipA/VipB tubules is triggered by ClpV. Here, we plan to unravel the basic working principle of the V. cholerae T6SS. We aim at providing experimental evidence for the function of VipA/VipB as contractile proteins and to identify novel interaction partners of ClpV and VipA/VipB, which might control tubule severing. Based on our recent findings showing a distinct cellular localization of T6SSs, we will employ immunofluorescence and immunoelectron microscopy to determine the subcellular organization of T6SSs and to position VipA/VipB tubules, ClpV and the exoprotein Hcp in a T6S assembly. Together these approaches will lead to an advanced mechanistic understanding of T6SSs. Our second main goal is to characterize the interaction of ClpV and VipA/VipB at the molecular level and to identify small compounds that inhibit T6SSs by affecting the interaction between ClpV and VipA/VipB tubules. We recently determined the crystal structure of the ClpV N-domain and identified its interaction site in the VipA/VipB tubule within the N-terminal region of VipB. We are aiming at determining the precise binding mode by co-crystallization and validating the structure by subsequent mutagenesis. A meanwhile established peptide binding assay sets the basis for establishing a medium-throughput ALPHA screen to identify small compounds that prevent the ClpV-VipA/VipB interaction. The potential of such molecules as antimicrobial agents will then be tested in vitro and in vivo.

VI 型分泌系统 (T6SS) 存在于许多致病性革兰氏阴性细菌中，并通过将效应子传递到靶细胞中来促进各种物种的毒力。 T6SS 的外蛋白（Hcp、VgrG）与带尾噬菌体的细胞穿刺装置的组件表现出结构相似性。因此，有人认为 T6SS 代表收缩注射机器，从而使用独特的机制将毒素靶向宿主细胞。我们可以通过切断 VipA/VipB 小管（T6SS 的保守且重要组成部分）来证明 AAA+ 伴侣 ClpV 作为 T6SS 的必需 ATP 酶。 VipA/VipB 管的结构表明它们的功能可能类似于噬菌体的尾鞘蛋白，通过收缩驱动 T6SS 外蛋白的喷射。我们认为 VipA/VipB 小管的这种构象变化是由 ClpV 触发的。在这里，我们计划揭开霍乱弧菌T6SS的基本工作原理。我们的目的是为 VipA/VipB 作为收缩蛋白的功能提供实验证据，并确定 ClpV 和 VipA/VipB 的新相互作用伙伴，它们可能控制肾小管切断。基于我们最近显示 T6SS 的独特细胞定位的发现，我们将采用免疫荧光和免疫电子显微镜来确定 T6SS 的亚细胞组织，并在 T6S 组装中定位 VipA/VipB 小管、ClpV 和外蛋白 Hcp。这些方法共同将带来对 T6SS 的高级机械理解。我们的第二个主要目标是在分子水平上表征 ClpV 和 VipA/VipB 的相互作用，并鉴定通过影响 ClpV 和 VipA/VipB 小管之间的相互作用来抑制 T6SS 的小化合物。我们最近确定了 ClpV N 结构域的晶体结构，并确定了其在 VipB N 末端区域内的 VipA/VipB 小管中的相互作用位点。我们的目标是通过共结晶确定精确的结合模式，并通过随后的诱变验证结构。同时建立的肽结合测定为建立中等通量 ALPHA 筛选奠定了基础，以鉴定阻止 ClpV-VipA/VipB 相互作用的小化合物。然后将在体外和体内测试此类分子作为抗菌剂的潜力。