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

将效应蛋白输送到宿主细胞的分泌系统是许多细菌病原体的基本毒力因子。新近发现的VI型分泌系统(T6SS)就是这样一个系统，它被证明对各种革兰氏阴性菌的细菌-宿主相互作用具有重要影响。我们将使用引起致命疾病类鼻疽的人和动物兼性细胞内病原体假性伯克霍尔德氏菌作为模式生物，研究T6S在细菌发病中的作用。假鼻疽杆菌建立感染的毒力策略是不明确的；然而，T6S已经成为假鼻疽杆菌的一个关键毒力因子。特别是，在细菌中发现的一种T6SS-5的失活，使其在通常致命的感染剂量下是无毒的，并且无法在小鼠肺组织中增殖。此外，已知T6SS-5是促进宿主细胞融合所必需的，这一过程促进了假鼻疽杆菌的细胞间传播和它们逃避细胞外免疫防御。然而，T6SS-5及其底物产生观察到的表型的机制尚不清楚。我们的长期目标是确定T6SS-5依赖的与宿主的相互作用的分子基础，这些相互作用有助于疾病的发生。我们先前在泰兰分枝杆菌中鉴定出VgrG-5是一种T6SS-5分泌蛋白，它是假鼻疽中VgrG-5的同源物。VgrG-5基因的特定截断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通过直接或间接的方式介导宿主细胞的融合，在假鼻疽杆菌的毒力中发挥关键作用。这项建议的目的是对假鼻疽杆菌VgrG-5在感染过程中的活性进行全面分析。这将通过追求以下特定目标来实现：(1)利用时间推移和免疫荧光显微镜确定T6SS-5激活的时空要求和VgrG-5在宿主细胞中的定位，(2)利用感染的巨噬细胞的RNA-Seq对VgrG-5的转录宿主反应进行全基因组调查，(3)确定VgrG-5的细胞靶标，并确定这种相互作用在假鼻疽杆菌细胞间传播和毒力中的相关性。本研究将从机制上对T6SS-5的功能和类鼻疽病的发病机制进行深入的研究。最后，我们希望更好地了解假单胞菌是如何抵消宿主清除的，将揭示抗感染药物的靶点。

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.