Host factors in Shigella flexneri infection

福氏志贺菌感染的宿主因素

• 批准号: 10197816
• 负责人: Marcia B Goldberg
• 金额: $ 49.13万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2023-02-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10197816
• 关键词: Actins; Acute; Acylation; Bacteria; Bacterial Proteins; Binding; Biochemical; C-terminal; Caspase; Cell Death; Cell Proliferation; Cell membrane; Cell physiology; Cells; Colon; Cytosol; Data; Diarrhea; Disease; Docking; Dysentery; Epithelial; Feedback; Funding; Genes; Genetic; Genetic Screening; Goals; Haploid Cells; Human; IQ motif containing GTPase activating protein 1; In Vitro; Infection; Inflammasome; Inflammation; Inflammatory Response; Innate Immune Response; Integration Host Factors; Intermediate Filaments; Investigation; Lead; Leucine-Rich Repeat; Maps; Membrane; Monomeric GTP-Binding Proteins; Needles; Nucleotides; Pathogenesis; Pathway interactions; Pore Proteins; Positioning Attribute; Process; Progress Reports; Protein Family; Protein Secretion; Proteins; Role; Scaffolding Protein; Shigella; Shigella flexneri; Signal Transduction; Testing; Type III Secretion System Pathway; Vacuole; Virulence; base; cell growth; cell motility; cytokine; design; human pathogen; macrophage; marenostrin; member; mutant; pathogen; prevent; protein activation; protein function; protein protein interaction; response; sensor; uptake

Shigella species cause diarrhea and dysentery in humans by infecting the colon. Pathogenesis depends on regulated interactions between the host and pathogen. Whereas identification and characterization of Shigella virulence proteins has advanced considerably, it has been more challenging to identify host proteins that participate in infection. During the current funding period, we defined the roles of several host proteins in S. flexneri pathogenesis and identified dozens of other host proteins in broad-based screens designed to discover host genes that promote S. flexneri infection. Our goal is to continue to build on these discoveries by conducting in depth investigations into how a two of these host proteins function in Shigella pathogenesis. 1. Analyze the topology, organization, and function of the translocon pore relevant to S. flexneri docking and effector translocation. We discovered that docking of the T3SS needle apparatus onto the membrane-embedded translocon pore depends on interaction of the pore protein IpaC with cellular intermediate filaments, yet pore formation per se is independent of this interaction. Based on these findings, we are now able to allow pore formation while preventing docking of bacteria, thereby enabling us to (1) map the topology, symmetry, and stoichioletry of the translocon pore proteins upon delivery by the T3SS, (2) define the relative positions of and interactions between the two translocon proteins in membrane-embedded helices, (3) test our postulate that C-terminal sequences in IpaC enter the interior of the pore, and (4) investigate how interaction of intermediate filaments with IpaC enables docking of the needle on the pore. 2. Determine the function of NLRP11 in responses of human macrophages to S. flexneri infection. Whereas some NLRP proteins are constituents of inflammasomes, NLRP11 has no known function. Our data show that NLRP11 is required for efficient cell death induced by S. flexneri infection and by cytosolic LPS (cLPS). cLPS triggers the activation of an inflammasome that contains caspases-4 and 5, for which no NLRP or other sensor molecule has been identified. Because LPS binds caspases-4 and 5 directly in vitro, it has been postulated that the cLPS inflammasome may not require an NLRP or sensor molecule. Based on our data, we hypothesize that NLRP11 functions as a sensor molecule for the cLPS inflammasome pathway. We will test this hypothesis using a combination of genetic and biochemical approaches to (1) determine the function of NLRP11 in macrophage responses to S. flexneri and cLPS, (2) determine whether acylation state of LPS, which modulates innate immune responses to S. flexneri, modulates NLRP11 recognition and/or signaling, and (3) define the breadth of human pathogens that trigger NLRP11-dependent responses.

志贺氏菌属通过感染结肠引起人类腹泻和痢疾。发病机制取决于 调节宿主和病原体之间的相互作用。鉴于志贺氏菌的鉴定和表征 毒力蛋白已经有了相当大的进步，识别宿主蛋白变得更具挑战性。 参与感染。在当前的资助期间，我们定义了几种宿主蛋白​​在金黄色葡萄球菌中的作用。 福氏菌发病机制，并在广泛的筛选中鉴定了数十种其他宿主蛋白，旨在发现 促进弗氏沙门氏菌感染的宿主基因。我们的目标是继续以这些发现为基础 深入研究其中两种宿主蛋白​​如何在志贺氏菌发病机制中发挥作用。 1. 分析与 S. flexneri 相关的易位子孔的拓扑、组织和功能 对接和效应器易位。我们发现将 T3SS 针装置对接到 膜嵌入易位子孔取决于孔蛋白 IpaC 与细胞的相互作用 中间丝，但孔的形成本身与这种相互作用无关。基于这些发现， 我们现在能够允许孔形成，同时防止细菌对接，从而使我们能够 (1) 绘制地图 T3SS 递送后易位子孔蛋白的拓扑结构、对称性和化学配比，(2) 定义 膜嵌入螺旋中两个易位子蛋白的相对位置和相互作用， (3) 测试我们的假设，即 IpaC 中的 C 端序列进入孔内部，以及 (4) 研究如何 中间丝与 IpaC 的相互作用使针能够停靠在孔上。 2.确定NLRP11在人类巨噬细胞对福氏链球菌感染的反应中的功能。 虽然一些 NLRP 蛋白是炎症小体的组成部分，但 NLRP11 的功能尚不清楚。我们的数据 显示 NLRP11 是福氏链球菌感染和胞质 LPS 诱导的有效细胞死亡所必需的 （cLPS）。 cLPS 触发包含 caspase-4 和 5 的炎症小体的激活，而 NLRP 对此无作用 或其他传感器分子已被识别。由于 LPS 在体外直接结合 caspase-4 和 5，因此具有 据推测，cLPS 炎性体可能不需要 NLRP 或传感器分子。基于我们的 根据数据，我们假设 NLRP11 充当 cLPS 炎性体途径的传感器分子。我们 将结合遗传和生化方法来检验这一假设：(1) 确定 NLRP11 在巨噬细胞对福氏链球菌和 cLPS 反应中的功能，(2) 确定 NLRP11 的酰化状态是否 LPS 可调节对弗氏链球菌的先天免疫反应，调节 NLRP11 识别和/或 (3) 定义触发 NLRP11 依赖性反应的人类病原体的范围。