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

宿主细胞骨架、细胞粘​​附和膜运输蛋白在 b 中的作用

• 批准号: 8830430
• 负责人: Matthew D Welch
• 金额: $ 23.53万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-02 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8830430
• 关键词: Actins; Bacteria; Cell Adhesion; Cell Adhesion Molecules; Cell Communication; Cell membrane; Cell-Cell Adhesion; Cells; Communication; Confocal Microscopy; Cytoskeleton; Cytosol; Diagnosis; Endocytosis; Fever; Gene Silencing; Health; Image; Immune response; Individual; Infection; Integration Host Factors; Invaded; Left; Length; Life; Life Cycle Stages; Listeria; Listeria monocytogenes; Listeriosis; Membrane; Membrane Protein Traffic; Molecular; Nature; Pathway interactions; Phagocytosis; Play; Process; Proteins; RNA Interference; Rickettsia; Role; Spottings; Staging; Testing; Time; Typhus; Vacuole; Virulence; Work; cell motility; cellular imaging; comparative; human disease; novel strategies; pathogen; protein function; research study

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.

描述（由申请人提供）：许多细菌性疾病，包括李斯特菌物种和立克群的斑点发烧组（SFG），入侵宿主细胞，脱离浸润液泡，然后在细胞质中生长，在那里动员宿主宿主肌动蛋白细胞骨骼骨骼为细胞内运动和细胞间扩散供电。这些细菌在细胞之间传播的能力而不离开细胞的范围，可以逃避体液免疫反应，这是导致其在斑点发烧疾病中毒力的关键因素（美国R. rickettsii和r parkeri）和李斯特氏病（ L.单核细胞增生）。尽管细胞间传播在感染和毒力中的重要性，但该过程是这些病原体细胞内生命周期中最糟糕的阶段。李斯特菌和SFG人力素的细胞间扩散发生在包括与供体细胞膜相撞的步骤中，突起形成，将突起的内在化，由受体细胞将液泡内部化到液泡中以及液泡逃脱。但是，我们不知道在此过程中的每个步骤中，这些病原体在宿主中利用了哪些细胞途径。在此处提出的探索性实验中，我们将测试总体假设，即李斯特菌和立克山的步骤既需要重叠和不同的宿主蛋白集，包括对皮质细胞骨骼功能，细胞粘附，细胞粘附，细胞粘附，重要的蛋白质集合。 吞噬作用和内吞作用。特别是，我们将回答两个问题。哪种宿主蛋白​​在物理或李斯特氏症上的功能很重要，是其一般或病原体的作用 具体的？此外，在每个宿主蛋白质的传播阶段，供体或受体细胞中是否需要它们？在AIM 1中，我们将执行平行的RNAi筛查，以180个宿主因素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，并评估基因沉默对Rickettsia和Listeria蔓延的影响。这种比较方法将揭示蛋白质和途径通常对病原体扩散以及对每种病原体特别重要的蛋白质和途径。在AIM 2中，我们将使用RNAi和Live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的突出形成中作用的特定假设。 并吞噬受体细胞，而吞噬作用和内吞作用蛋白作用于突出吞噬。此外，我们将通过确定其在扩散过程中确定其定位并测试其活性是否在供体或食物细胞中确定其活性来研究其功能。通过这些探索性实验，我们将开发一个框架，以理解病原体细胞传播的关键机制，这将为对该过程的机械理解奠定基础。这些研究可能揭示了宿主 - 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。