Role of type VI secretion in Burkholderia pseudomallei virulence and host interaction

假性伯克霍尔德菌是类鼻疽病的病原，是一种严重的侵袭性人畜感染。假芽孢杆菌的基因组包含6个编码表观VI型分泌系统（T6SS）的基因簇，其功能尚不清楚。t6ss是最近被描述的蛋白质输出机器，它将底物释放到细胞外环境和宿主细胞的细胞质中。新出现的数据表明，t6ss在许多致病细菌和共生细菌与宿主的相互作用中起着至关重要的作用。本项目的第一个目的是系统分析假芽孢杆菌各预测T6SS在毒力中的作用。我们将从产生能使每个T6SS失活的帧内突变菌株开始。这些菌株将在小鼠急性肺炎类鼻疽模型中分析毒力。二是全面研究在假芽孢杆菌发病机制中发挥明确作用的t6ss的作用机制。为此，将通过细菌表型微阵列检查给定T6SS对细菌细胞过程的影响。一系列细胞培养感染试验将用于测量给定T6SS对相关宿主细胞类型的细胞毒性、入侵、复制和吞噬体逃逸的贡献。此外，我们将采用微阵列方法来表征全球t6s依赖性对宿主细胞反应的影响。本文提出的实验将为T6S影响假芽孢杆菌与其宿主相互作用的机制提供重要的新见解。该项目也将成为了解假芽孢杆菌发病机制基本方面的关键一步。

Burkholderia pseudomallei is the etiological agent of melioidosis, a serious invasive infection of humans and animals. The genome of B. pseudomallei harbours six gene clusters that encode apparent type VI secretion systems (T6SS) whose function is unknown. T6SSs are recently described protein export machines that release substrates into the extracellular milieu and into the cytosol of host cells. Emerging data demonstrate a crucial role for T6SSs in the interactions of many pathogenic and symbiotic bacteria with their hosts. The first aim of this project is to conduct a systematic analysis of the role of each predicted T6SS of B. pseudomallei in virulence. We will begin by generating strains bearing in-frame mutations that inactivate each T6SS. These strains will be analyzed for virulence in a murine model of acute pneumonic melioidosis. The second aim is to comprehensively investigate the mechanism-ofaction of those T6SSs that play demonstrable roles in B. pseudomallei pathogenesis. To this end the effect of a given T6SS on bacterial cellular processes will be examined via bacterial phenotype microarrays. A series of cell culture infection assays will be used to measure the contribution of a given T6SS to cytotoxicity, invasion, replication and phagosomal escape within relevant host cell types. In addition, we will pursue a microarray approach to characterize global T6S-dependent effects on host cell response. The experiments proposed herein will provide important new insights into the mechanism by which T6S influences the interaction between B. pseudomallei and its host. This project will also serve as a crucial step towards understanding basic aspects of B. pseudomallei pathogenesis.