Mechanism of type VI secretion system 5-dependent intercellular spread of Burkholderia pseudomallei

将效应蛋白递送到宿主细胞中的分泌系统是许多细菌病原体中的重要毒力因子。最近发现的VI型分泌系统（T6 SS）就是这样一个系统，其显示出严重影响各种革兰氏阴性人类病原体的细菌-宿主相互作用。我们将使用类鼻疽伯克霍尔德氏菌，一种兼性细胞内的人类和动物病原体，导致危及生命的疾病类鼻疽，作为模式生物的T6 S在细菌发病机制中的作用的研究。B.然而，T6 S已经成为B中的关键毒力因子。假鼻疽特别是，在细菌中发现的T6 SS之一T6 SS-5的失活使其在通常致死的感染剂量下无毒，并且不能在鼠肺组织中增殖。此外，已知需要T6 SS-5来促进宿主细胞的融合，这是促进B的细胞间扩散的过程。类鼻疽和它们逃避细胞外免疫防御。然而，T6 SS-5及其底物引起观察到的表型的机制尚不清楚。我们的长期目标是确定T6 SS-5依赖性与宿主相互作用的分子基础，从而导致疾病。我们先前鉴定VgrG-5为B中的T6 SS-5分泌蛋白。thailandensis，它是B中VgrG-5的同源物。假鼻疽VgrG-5中的特定截短和氨基酸缺失导致宿主细胞融合缺陷。我们的中心假设是VgrG-5在B的毒力中起关键作用。通过以直接或间接的方式介导宿主细胞的融合来治疗类鼻疽。本提案的目的是对B进行全面分析。假鼻疽VgrG-5活性。这将通过实现以下具体目标来实现：（1）使用时间推移和免疫荧光显微术确定T6 SS-5活化的时空要求和VgrG-5在宿主细胞内的定位，（2）利用感染的巨噬细胞的RNA-Seq进行对VgrG-5的转录宿主应答的全基因组调查，和（3）确定VgrG-5的细胞靶标。5并确定这种相互作用在B的细胞间传播和毒力中的相关性。假鼻疽本研究将为阐明T6 SS-5的功能和类鼻疽的发病机制提供理论依据。最后，我们期望能更好地理解B.假鼻疽抵抗宿主的清除将揭示抗感染剂的靶点。

Secretion systems that deliver effector proteins into host cells are essential virulence factors in a number of bacterial pathogens. The recently discovered type VI secretion system (T6SS) is one such system, which was shown to critically affect the bacteria-host interaction of various Gram negative human pathogens. We will use Burkholderia pseudomallei, a facultative intracellular human and animal pathogen that causes the life-threatening disease melioidosis, as a model organism for the study of the role of T6S in bacterial pathogenesis. The virulence strategies of B. pseudomallei to establish an infection are poorly defined; T6S however, has emerged as a key virulence factor in B. pseudomallei. In particular, the inactivation of one of the T6SSs found in the bacterium, T6SS-5, renders it avirulent at a usually lethal infection dose and unable to proliferate in murine lung tissue. Further, it is known that the T6SS-5 is required to promote the fusion of host cells, a process that facilitates intercellular spread of B. pseudomallei and their escape from extracellular immune defenses. However, the mechanism of T6SS-5 and its substrates that give rise to the observed phenotypes are unknown. Our long-term goal is to define the molecular basis of T6SS-5-dependent interactions with the host that contribute to disease. We previously identified VgrG-5 as a T6SS-5-secreted protein in B. thailandensis, which is a homolog of VgrG-5 in B. pseudomallei. Specific truncations of and amino acid deletions in VgrG-5 result in a host cell fusion defect. Our central hypothesis is that VgrG-5 plays a key role in the virulence of B. pseudomallei by mediating the fusion of host cells in a direct or indirect manner. The objective of this proposal is to perform a comprehensive analysis of B. pseudomallei VgrG-5 activity during infection. This will be accomplished by pursuing the following specific aims: (1) Define the spatiotemporal requirements of T6SS-5 activation and localization of VgrG-5 within host cells using time lapse and immunofluorescence microscopy, (2) Performing a genome-wide survey of the transcriptional host response to VgrG-5 utilizing RNA-Seq of infected macrophages and (3) Determine the cellular target of VgrG-5 and define the relevance of this interaction in intercellular spread and virulence of B. pseudomallei. This study will provide a mechanistic insight into the function of T6SS-5 and the pathogenesis of melioidosis. Finally, we expect that a better understanding of how B. pseudomallei counteracts clearance by the host will reveal targets for anti-infective agents.