Mechanism of type VI protein secretion

VI型分泌系统(T6SS)存在于许多致病革兰氏阴性菌中，通过将效应物输送到靶细胞而在不同物种中发挥毒力。T6SSs(Hcp，VgrG)的外源蛋白在结构上与尾随噬菌体的细胞穿透装置的组成成分具有相似的结构。因此，有人认为T6SS代表收缩注射机制，从而使用一种独特的机制将毒素靶向宿主细胞。我们可以通过切断VIPA/VipB小管来证明AAA+伴侣ClpV是T6SS的一个必不可少的ATPase。VIPA/VipB小管的结构表明，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外源蛋白的排出。我们认为这种VIPA/VipB小管的构象变化是由ClpV触发的。在这里，我们计划解开霍乱弧菌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，并寻找可能控制小管切断的ClpV和VIPA/VipB的新的相互作用伙伴。基于我们最近发现的T6SS的明显细胞定位，我们将使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。综合起来，这些方法将导致对T6SS的机械理解的深入。我们的第二个主要目标是在分子水平上表征ClpV和VIPA/VipB之间的相互作用，并通过影响ClpV和VIPA/VipB之间的相互作用来识别抑制T6SS的小分子化合物。我们最近确定了ClpV N-结构域的晶体结构，并在VipB的N-末端区域内的VIPA/VipB小管中确定了它的相互作用位置。我们的目标是通过共结晶来确定准确的结合模式，并通过随后的突变来验证结构。同时，建立的多肽结合试验为建立中通量Alpha筛查奠定了基础，以识别阻止ClpV-VIPA/VipB相互作用的小化合物。然后，这些分子作为抗菌剂的潜力将在体外和体内进行测试。

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.