Roles for host cytoskeletal, cell adhesion and membrane trafficking proteins in b

描述（由申请人提供）：许多细菌病原体，包括李斯特菌和立克次氏体的斑点热组（SFG），侵入宿主细胞，从入侵液泡中逃脱，然后在细胞质中生长，在那里它们动员宿主肌动蛋白细胞骨架，为细胞内运动和细胞间传播提供动力。这些细菌在细胞之间传播而不离开细胞范围的能力使其能够逃避体液免疫反应，并且是它们在斑点热疾病（美国的立克次体和帕克氏体）和李斯特菌病（单核增生李斯特菌）中的毒性的关键因素。尽管细胞间传播在感染和毒力中很重要，但这一过程是这些病原体细胞内生命周期中最不为人所知的阶段。李斯特菌和SFG立克次体的细胞间传播发生的步骤包括：与供体细胞膜碰撞，形成突起，突起被受体细胞内化到液泡中，以及液泡逃逸。然而，我们不知道这些病原体在这个过程的每一步都利用了宿主的哪些细胞途径。在这里提出的探索性实验中，我们将测试李斯特菌和立克次体传播的步骤需要重叠和不同的宿主蛋白集的总体假设，包括那些对皮质细胞骨架功能，细胞粘附，

DESCRIPTION (provided by applicant): Many bacterial athogens, including Listeria species and the spotted fever group (SFG) of Rickettsia species, invade host cells, escape from the invasion vacuole, and then grow in the cytosol, where they mobilize the host actin cytoskeleton to power intracellular motility and cell-to-cell spread. The ability of these bacteria to spread between cells without leaving the confines of the cell enables evasion of the humoral immune response, and is a key contributor to their virulence in spotted fever illness (R. rickettsii and R parkeri in the U.S.) and listeriosis (L. monocytogenes). Despite the importance of cell-to-cell spread in infection and virulence, this process is the most poorly understood stage in the intracellular life cycle of these pathogens. Cell-to-cell spread of Listeria and SFG Rickettsia occurs in steps that include collision with the donor cell membrane, formation of a protrusion, internalization of the protrusion into a vacuole by a recipient cell, and vacuole escape. However, we do not know which cellular pathways in the host are exploited by these pathogens at each step in this process. In the exploratory experiments proposed here, we will test the overall hypothesis that the steps of Listeria and Rickettsia spread require both overlapping and distinct sets of host proteins, including those important for cortical cytoskeletal function, cell adhesion, phagocytosis and endocytosis. In particular, we will answer two questions. Which host proteins are functionally important for Rickettsia or Listeria spread, and is their role general or pathogen specific? Moreover, at which stage of spread does each host protein act, and are they required in the donor or recipient cell? In Aim 1, we will carry out parallel RNAi screens, targeting 180 host factors important for cortical cytoskeleton function, cell-cell adhesion, phagocytosis and endocytosis, and evaluate the impact of gene silencing on both Rickettsia and Listeria spread. This comparative approach will reveal proteins and pathways that are generally important for pathogen spread, as well as those that are specifically important for each pathogen. In Aim 2, we will use a combination of RNAi and live cell imaging to test the specific hypotheses that cortical cytoskeleton and cell-cell adhesion proteins act in protrusion formation in the donor cell and engulfment into the recipient cell, whereas phagocytosis and endocytosis proteins act in protrusion engulfment. Additionally, we will examine how each factor functions by determining its localization during spread and testing whether its activity is required in the donor or recipiet cell. Through these exploratory experiments, we will develop a framework for understanding crucial mechanisms of pathogen cell- to-cell spread that will set the stage for a more mechanistic understanding of this process. These studies may reveal new mechanisms of host-pathogen interactions, new approaches for diagnosing and treating infections, and new principles of cell-cell interaction and communication in uninfected cells.