Genetic Analysis of Legionella Phagosome Trafficking

嗜肺军团菌是一种叫做军团病的严重肺炎的病原体。军团菌在吞噬细胞内复制的能力是宿主发病机制的核心，需要一种称为Dot/Icm的特殊分泌系统。该项目的重点是确定Dot/Icm系统如何使军团菌产生支持复制的细胞内液泡。为此，我们已经证明这一过程涉及宿主内质网衍生囊泡的颠覆，军团菌利用这些囊泡将其最初占据的质膜衍生细胞器重塑为类似内质网的液泡。通过Dot/Icm系统传递到宿主细胞的蛋白质促进了这一膜运输途径。在过去的资助期内，我们已经确定了针对宿主膜运输调节因子Rab1的多种细菌蛋白。为了了解这些蛋白如何控制膜运输和促进液泡的生物发生，从而允许军团菌在细胞内生长，我们将研究这些蛋白在军团菌感染宿主细胞时的功能。具体而言，我们将确定这些蛋白的生化活性在感染过程中如何在空间和时间上被调节，以协调Rab1在含有军团菌的液泡上的循环，确定Rab1在含有军团菌的液泡上的激活如何促进内质网来源的囊泡的招募和融合，并确定调节磷脂酰肌醇4-磷酸的宿主蛋白在液泡成熟中的作用。

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. The ability of Legionella to replicate inside of phagocytic cells is central to host pathogenesis and requires a specialized secretion system called Dot/Icm. The focus of this project has been to determine how the Dot/Icm system enables Legionella to create an intracellular vacuole that supports replication. Towards this end, we have demonstrated that this process involves the subversion of host vesicles derived from the endoplasmic reticulum, which are used by Legionella to remodel the plasma membrane-derived organelle it occupies initially into a vacuole that resembles the endoplasmic reticulum. Proteins delivered into host cells by the Dot/Icm system promote this membrane transport pathway. Over the past funding period we have identified multiple bacterial proteins that target the host membrane transport regulator Rab1. To understand how these proteins control membrane transport and promote biogenesis of a vacuole that permits Legionella intracellular growth, we will investigate the function of these proteins during infection of host cells by Legionella. Specifically, we will determine how the biochemical activities of these proteins are regulated spatially and temporally during infection to coordinate the cycling of Rab1 on vacuoles containing Legionella, determine how Rab1 activation on the vacuole containing Legionella can promote the recruitment and fusion of endoplasmic reticulum-derived vesicles, and determine the role host proteins that regulate phosphatidylinositol 4-phosphate play in vacuole maturation.