Genetic Analysis of Legionella Phagosome Trafficking

嗜肺军团菌是一种名为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的核心，需要一种称为Dot/ICM的特殊分泌系统。该项目的重点一直是确定Dot/ICM系统如何使军团菌能够创建支持复制的细胞内空泡。为此，我们已经证明，这一过程涉及到颠覆来自内质网的宿主小泡，军团菌利用这些小泡将最初占据的质膜衍生细胞器重塑为类似内质网的液泡。由Dot/ICM系统运送到宿主细胞的蛋白质促进了这一膜转运途径。在过去的资助期间，我们已经确定了多种以宿主膜运输调节因子Rab1为靶标的细菌蛋白。为了了解这些蛋白如何控制膜运输并促进允许军团菌在细胞内生长的空泡的生物发生，我们将研究这些蛋白在军团菌感染宿主细胞过程中的功能。具体地说，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进行调节，以协调含有军团菌的空泡上Rab1的循环，确定在含有军团菌的空泡上激活Rab1如何促进内质网衍生小泡的招募和融合，并确定调节磷脂酰肌醇4-磷酸的宿主蛋白在空泡成熟中所起的作用。

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.