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。而志贺氏菌的鉴定和特征 毒力蛋白的研究已经取得了相当大的进展，但是鉴定 参与感染。在目前的资助期间，我们确定了几种宿主蛋白在S。 并在基础广泛的筛选中鉴定了数十种其他宿主蛋白， 宿主基因促进S.弗氏菌感染我们的目标是继续在这些发现的基础上， 深入研究这些宿主蛋白中的两种在志贺氏菌发病机制中的作用。 1.分析了S.福氏 对接和效应子易位。我们发现，将T3 SS针装置对接到 膜包埋的易位子孔依赖于孔蛋白IpaC与细胞的相互作用， 中间丝，但孔的形成本身是独立的这种相互作用。根据这些发现， 我们现在能够在防止细菌对接的同时允许孔形成，从而使我们能够（1）绘制 在通过T3 SS递送时，易位孔蛋白的拓扑结构、对称性和化学计量学，（2）定义 膜包埋螺旋中两种转位子蛋白的相对位置和相互作用， (3)测试我们假设IpaC中的C-末端序列进入孔的内部，和（4）研究如何 中间丝与IpaC的相互作用使得针能够对接在孔上。 2.确定NLRP 11在人巨噬细胞对S.弗氏菌感染 尽管一些NLRP蛋白是炎性小体的成分，但NLRP 11没有已知的功能。我们的数据 显示NLRP 11是由S.福氏杆菌感染和胞质LPS （cLPS）。cLPS触发含有半胱天冬酶-4和5的炎性小体的激活，对于这些炎性小体，没有NLRP 或其他传感器分子。由于LPS在体外直接结合半胱天冬酶-4和5， 假设cLPS炎性体可能不需要NLRP或传感器分子。基于我们 数据，我们假设NLRP 11作为cLPS炎性体通路的传感器分子发挥作用。我们 将使用遗传和生物化学方法的组合来测试这一假设，以（1）确定 NLRP 11在巨噬细胞对S. flexneri和cLPS，（2）确定是否酰化状态 LPS调节对S.弗氏杆菌，调节NLRP 11识别和/或 信号传导，和（3）定义触发NLRP 11依赖性应答的人类病原体的宽度。