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，包括李斯特菌属和立克次氏体属的斑点热群（SFG），侵入宿主细胞，从侵入空泡中逃逸，然后在胞质溶胶中生长，在胞质溶胶中，它们动员宿主肌动蛋白细胞骨架，为细胞内运动和细胞间扩散提供动力。这些细菌在细胞之间传播而不离开细胞范围的能力使得能够逃避体液免疫应答，并且是它们在斑点热疾病中的毒力的关键因素（R.美国的立克次氏体和帕克氏体）和黑胫病（L.单核细胞增多症）。尽管细胞间传播在感染和毒力中的重要性，但这一过程是这些病原体细胞内生命周期中最不了解的阶段。李斯特菌和SFG立克次氏体的细胞间传播发生在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的步骤中。然而，我们不知道在这个过程的每一步，这些病原体都利用了宿主中的哪些细胞途径。在这里提出的探索性实验中，我们将测试总体假设，即李斯特菌和立克次氏体传播的步骤需要重叠和不同的宿主蛋白质组，包括对皮质细胞骨架功能，细胞粘附， 吞噬作用和内吞作用。我们将特别回答两个问题。哪些宿主蛋白对立克次体或李斯特菌的传播具有重要的功能，它们的作用是一般性的还是病原性的 具体的？此外，每种宿主蛋白质在扩散的哪个阶段起作用，供体细胞或受体细胞是否需要它们？在目标1中，我们将进行平行RNAi筛选，靶向180个对皮质细胞骨架功能、细胞-细胞粘附、吞噬作用和内吞作用重要的宿主因子，并评估基因沉默对立克次体和李斯特菌传播的影响。这种比较方法将揭示通常对病原体传播重要的蛋白质和途径，以及对每种病原体特别重要的蛋白质和途径。在目标2中，我们将使用RNAi和活细胞成像的组合来测试皮质细胞骨架和细胞-细胞粘附蛋白在供体细胞中突起形成中起作用的特定假设 吞噬进入受体细胞，而吞噬作用和内吞作用蛋白在突起吞噬中起作用。此外，我们将通过确定其在扩散过程中的定位和测试其活性是否需要在供体或受体细胞中来检查每个因子的功能。通过这些探索性实验，我们将开发一个框架来理解病原体细胞到细胞传播的关键机制，这将为更机械地理解这一过程奠定基础。这些研究可能揭示宿主-病原体相互作用的新机制，诊断和治疗感染的新方法，以及未感染细胞中细胞-细胞相互作用和通信的新原理。

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.