Molecular Mechanism of cell-cell spread of Listeria in polarized epithelial cells

李斯特菌在极化上皮细胞中细胞间传播的分子机制

• 批准号: 7985826
• 负责人: KEITH Patrick IRETON
• 金额: $ 27.24万
• 依托单位: UNIVERSITY OF OTAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7985826
• 关键词: Actins; Adaptor Signaling Protein; Address; Affect; Animal Model; Apical; Bacteria; Bacterial Proteins; Binding; Cell Line; Cell membrane; Cells; Cellular biology; Cytoplasm; Cytoskeletal Proteins; Cytosol; DH Domain; Data; Defect; Development; Disease; Drug Delivery Systems; ERM protein; Enterocytes; Environment; Epithelial Cells; Epithelium; Evaluation; F-Actin; Gastroenteritis; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Hepatocyte; Human; Immune system; Immunofluorescence Microscopy; In Vitro; Infection; Intercellular Junctions; Intestines; Laboratories; Ligands; Listeria; Listeria monocytogenes; Listeriosis; Mammalian Cell; Mediating; Membrane; Membrane Proteins; Meningitis; Microbiology; Molecular; Molecular Biology; Morphology; Mus; Mutation; Phosphorylation; Play; Positioning Attribute; Probability; Process; Proteins; RNA Interference; Reagent; Research; Role; Scaffolding Protein; Site; Staging; Systemic disease; Tail; Testing; Virulence; Western Blotting; Work; abortion; cell motility; cell type; ezrin; foodborne; genetic regulatory protein; in vivo; mortality; pathogen; polarized cell; polymerization; public health relevance; receptor; research study; therapeutic development

DESCRIPTION (provided by applicant): Listeria monocytogenes is a food-borne, intracellular pathogen that causes gastroenteritis, abortions, or meningitis with a high mortality rate. Critical for Listeria virulence is its ability to replicate within mammalian cells and spread from one cell to another through an actin-dependent motility process that allows bacteria to remain within the protective environment of the host cytosol. Cell-cell spread of Listeria occurs in a variety of host cell types, including intestinal epithelial cells (enterocytes). Enterocytes are the first cell type infected by Listeria, and consequently play a key role in the onset of infection. Cell-cell spread is initiated by the bacterial surface protein ActA, which stimulates the formation of F-actin 'comet tails' that propel Listeria through the cytoplasm. Rocketing bacteria then contact the host cell plasma membrane and induce the formation of Listeria-containing protrusions that are engulfed by neighboring cells. ActA-mediated actin polymerization is well understood. However, little is known about the molecular mechanism of protrusion formation in enterocytes or other polarized epithelia. Specifically, it remains unclear whether bacterial proteins exist that function after ActA to directly control protrusion formation. Importantly, our preliminary data indicate that the secreted Listeria virulence protein InlC is needed for efficient protrusion formation and cell-cell spread in a polarized enterocyte cell line. InlC promotes bacterial protrusion generation by antagonizing the human adaptor protein Tuba. The precise mechanism by which InlC and Tuba control Listeria spreading remains unclear. As Tuba is a scaffolding protein, it likely affects spreading by interacting with one or more human ligands. The long-term goal of our work is to elucidate the molecular mechanism by which InlC promotes spreading of Listeria in polarized epithelial cells. The overall objective of this application is to identify human ligands of Tuba that control protrusion formation by Listeria. In addition, we aim to understand how these Tuba ligands are regulated by InlC. Our central hypothesis is that InlC controls protrusion formation by affecting Tuba and the activity and/or localization of one or more of Tuba's human ligands. The Specific Aims in this application, outlined below, are directed towards understanding how InlC, Tuba, and the ligands N-WASP, Cdc42, and ERM proteins control cell-cell spread. AIM 1. Determine the role of the Tuba ligands N-WASP and Cdc42 in Listeria protrusion formation, and elucidate how these host proteins are controlled by InlC. AIM 2. Determine if ERM proteins act together with Tuba to control bacterial spreading. AIM 3. Assess the role of InlC/Tuba interaction in virulence. The above objectives will be addressed through a variety of approaches, including immunofluorescence microscopy, evaluation of ERM protein phosphorylation through Western blotting, RNA interference to investigate the function of Tuba and its ligands in spreading, and virulence studies using mice. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes is a food-borne, intracellular bacterium that causes serious illnesses (listeriosis) leading to meningitis or abortion. Listeria evades the humoral immune system by spreading from one human cell to another, while remaining within the protective environment of the host cytosol. Epithelial cells lining the intestinal lumen (enterocytes) are the initial site of Listeria infection, and consequently play a critical role in the development of disease. The goal of this research is to identify bacterial and human components that mediate Listeria spreading in an enterocyte cell line. These bacterial and host proteins may be suitable as targets for drugs that limit Listeria infection.

描述(申请人提供)：单核细胞增多性李斯特菌是一种食源性细胞内病原体，可引起胃肠炎、流产或脑膜炎，死亡率高。李斯特菌毒力的关键是它在哺乳动物细胞内复制的能力，并通过依赖肌动蛋白的运动过程从一个细胞传播到另一个细胞，这一过程允许细菌留在宿主细胞质的保护环境中。李斯特菌的细胞-细胞传播发生在多种宿主细胞类型中，包括肠道上皮细胞(肠细胞)。肠上皮细胞是李斯特菌感染的第一个细胞类型，因此在感染的发生中起着关键作用。细胞-细胞扩散是由细菌表面蛋白ActA启动的，它刺激F-肌动蛋白“彗星尾巴”的形成，推动李斯特菌穿过细胞质。然后，火箭细菌接触宿主细胞质膜，诱导形成含有李斯特菌的突起，这些突起被邻近的细胞吞噬。ActA介导的肌动蛋白聚合是众所周知的。然而，对肠细胞或其他极化上皮中突起形成的分子机制知之甚少。具体地说，目前尚不清楚细菌蛋白是否存在在ActA之后直接控制突起形成的功能。重要的是，我们的初步数据表明，分泌的李斯特菌毒力蛋白InlC是在极化的肠细胞系中有效地形成突起和细胞-细胞扩散所必需的。InlC通过拮抗人类接头蛋白Tuba来促进细菌突起的产生。InlC和Tuba控制李斯特菌传播的确切机制尚不清楚。由于Tuba是一种支架蛋白，它可能通过与一个或多个人类配体相互作用来影响传播。我们工作的长期目标是阐明InlC促进李斯特菌在极化上皮细胞中传播的分子机制。这项应用的总体目标是确定控制李斯特菌突起形成的Tuba的人类配体。此外，我们的目标是了解这些Tuba配体是如何受到InlC的调控的。我们的中心假设是，InlC通过影响Tuba和Tuba的一个或多个人类配体的活性和/或定位来控制突起的形成。本申请的具体目的如下所述，旨在了解InlC、Tuba及其配体N-WASP、CDC42和ERM蛋白如何控制细胞间的扩散。目的1.确定TUBA配体N-WASP和CDC42在李斯特菌突起形成中的作用，并阐明这些宿主蛋白是如何受InlC调控的。目的2.确定ERM蛋白是否与Tuba共同作用以控制细菌传播。目的3.评估InlC/Tuba相互作用在毒力中的作用。上述目标将通过多种方法实现，包括免疫荧光显微镜、通过Western blotting评估ERM蛋白磷酸化、RNA干扰以研究Tuba及其配体在传播中的功能，以及使用小鼠进行毒力研究。 与公共卫生相关：单核细胞增生性李斯特菌是一种食源性细胞内细菌，可引起导致脑膜炎或流产的严重疾病(李斯特菌病)。李斯特菌通过从一个人类细胞传播到另一个细胞，同时留在宿主细胞质的保护环境中，从而逃避体液免疫系统。肠腔上皮细胞(肠细胞)是李斯特菌感染的起始部位，因此在疾病的发展中起着关键作用。这项研究的目标是确定介导李斯特菌在肠细胞系中传播的细菌和人类成分。这些细菌和宿主蛋白可能适合作为限制李斯特菌感染的药物的靶标。