The Role of TamAB in Salmonella Pathogenesis

摘要 鼠伤寒沙门氏菌是一种主要的食源性病原体，在巨噬细胞内繁殖， 导致危及生命的疾病。我们研究的长期目标是了解 沙门氏菌绕过宿主免疫系统致病的分子机制。 细菌的外膜是免疫系统的界面。外膜β- 桶蛋白通常由Bam复合体组装。Bam的同源物，TamAB 易位模块在变形杆菌中高度保守。最初是作为一个系统提出的 为了组装贝塔桶蛋白的子集，所谓的自动转运蛋白，实际的 TamAB的功能尚不清楚。我们发现TamAB是一个以前未被识别的成员 并在沙门氏菌在巨噬细胞中复制时被诱导，表明 这一系统对毒力很重要。事实上，我们的初步数据有力地支持了我们的初选 在应激条件下，TamAB被诱导辅助Bam功能的假说 噬菌体。我们的第二个假设是TamAB是正确组装 在吞噬小体中的条件下，外膜中的特定蛋白质，以及 这些蛋白质的减少或丢失会减弱沙门氏菌的毒力。在目标1中，我们将确定 外膜成分使用质谱仪，并监测包膜应力的表达 反应系统，以确定TamAB丢失引起的生化和调节效应， 从而识别直接或间接需要TamAB才能正确组装的蛋白质。我们还将 分离在体外抑制外膜通透性缺陷的突变 失去TamAB加剧了这一局面。在目标2中，我们将描述已识别的基因座及其在 巨噬细胞的存活和毒力。已确定的因素在巨噬细胞存活中的总体作用 将通过与TamAB的遗传相互作用来揭示。了解TamAB的角色将为我们提供信息 关于吞噬体内导致外膜装配应力的条件和 沙门氏菌用来对抗它们的机制。确定受基因缺失影响的特定蛋白质 TamAB还确定了对沙门氏菌致病至关重要的其他毒力因子。

ABSTRACT Salmonella Typhimurium is a major food-borne pathogen that propagates within macrophages, leading to life-threatening disease. The long-term objectives of our research are to understand the molecular mechanisms by which Salmonella circumvents the host immune system to cause disease. The outer membrane of the bacterium is the interface with the immune system. Outer membrane beta- barrel proteins are normally assembled by the Bam complex. A homolog of Bam, the TamAB translocation module is highly conserved in the Gammaproteobacteria. Originally proposed as a system for assembly of a subset of beta-barrel proteins, the so-called autotransporter proteins, the actual function of TamAB is unknown. We have discovered that tamAB is a previously unrecognized member of the PhoPQ regulon and is induced when Salmonella is replicating in macrophages, suggesting that this system is important for virulence. Indeed, our preliminary data strongly support our primary hypothesis that TamAB is induced to assist Bam function under the stressful conditions in the phagosome. Our secondary hypothesis is that TamAB is required for proper assembly of particular proteins in the outer membrane under the conditions in the phagosome, and that decreases or loss of these proteins attenuate Salmonella virulence. In Aim 1, we will determine outer membrane composition using mass spectrometry, and monitor expression of envelope stress response systems, to determine the biochemical and regulatory effects caused by loss of TamAB, thereby identifying proteins that directly or indirectly require TamAB for proper assembly. We will also isolate mutations that suppress in vitro defects in outer membrane permeability conferred or exacerbated by loss of TamAB. In Aim 2, we will characterize identified loci and their role in macrophage survival and virulence. The overall role of the identified factors in macrophage survival will be revealed by genetic interactions with tamAB. Understanding the roles of TamAB will inform us about the conditions in the phagosome leading to outer membrane assembly stress and the mechanisms used by Salmonella to combat them. Identifying the specific proteins affected by loss of TamAB also identifies additional virulence factors critical for Salmonella pathogenesis.