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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

基本信息

批准号：
8513124
负责人：
KEITH Patrick IRETON
金额：
$ 22.72万
依托单位：
UNIVERSITY OF OTAGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-08-15 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8513124
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 Targeting 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 N-WASP protein 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 cdc42 GTP-Binding Protein cell motility cell type ezrin foodborne genetic regulatory protein in vivo mortality pathogen polarized cell polymerization public health relevance receptor research study therapeutic development

项目摘要

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（由申请方提供）：单核细胞增生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其在哺乳动物细胞内复制并通过肌动蛋白依赖性运动过程从一个细胞传播到另一个细胞的能力，该过程允许细菌保持在宿主胞质溶胶的保护环境内。李斯特菌的细胞-细胞传播发生在多种宿主细胞类型中，包括肠上皮细胞（肠上皮细胞）。肠细胞是李斯特菌感染的第一种细胞类型，因此在感染的发生中起关键作用。细胞间的传播是由细菌表面蛋白ActA启动的，它刺激F-肌动蛋白“彗星尾”的形成，推动李斯特菌穿过细胞质。火箭细菌然后接触宿主细胞质膜并诱导形成被邻近细胞吞噬的含有李斯特菌的突起。ActA介导的肌动蛋白聚合是很好理解的。然而，对肠上皮细胞或其他极化上皮细胞突起形成的分子机制知之甚少。具体而言，目前尚不清楚是否存在细菌蛋白质，其在ActA之后发挥作用以直接控制突起形成。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是极化肠上皮细胞系中有效突起形成和细胞-细胞扩散所必需的。InlC通过拮抗人衔接蛋白Tuba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，它可能通过与一种或多种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。本申请的总体目标是鉴定控制李斯特菌突起形成的Tuba的人配体。此外，我们的目标是了解这些Tuba配体是如何调节InlC。我们的中心假设是InlC通过影响Tuba和Tuba的一种或多种人配体的活性和/或定位来控制突起形成。下文概述的本申请中的具体目的涉及理解InlC、Tuba和配体N-WASP、Cdc 42和ERM蛋白如何控制细胞-细胞扩散。 AIM 1.确定Tuba配体N-WASP和Cdc 42在李斯特菌突起形成中的作用，并阐明InlC如何控制这些宿主蛋白。AIM 2.确定ERM蛋白是否与Tuba一起控制细菌传播。 AIM 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来解决，包括免疫荧光显微镜，通过Western印迹法评价ERM蛋白磷酸化，RNA干扰以研究Tuba及其配体在传播中的功能，以及使用小鼠的毒力研究。公共卫生相关性：单核细胞增生李斯特菌是一种食源性细胞内细菌，可引起严重疾病（脑膜炎），导致脑膜炎或流产。李斯特菌通过从一个人体细胞传播到另一个细胞来逃避体液免疫系统，同时保持在宿主胞质溶胶的保护性环境中。肠腔的上皮细胞（肠上皮细胞）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。本研究的目的是确定介导李斯特菌在肠上皮细胞系中传播的细菌和人体成分。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点。