Role of the Listeria protein InlC and its host ligand Tuba in cell-cell spread

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，能够引起胃肠炎、脑膜炎和流产。李斯特菌毒力的关键是其在哺乳动物细胞内复制的能力，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，该过程允许细菌保持在宿主胞质溶胶的保护性环境中。细胞-细胞传播是由细菌表面蛋白ActA启动的，ActA刺激F-肌动蛋白“彗星尾巴”的形成，推动李斯特菌通过细胞质。火箭细菌接触宿主细胞质膜，并诱导形成被邻近细胞吞噬的含李斯特菌的突起。虽然ActA介导的肌动蛋白聚合是很好的理解，它仍然不清楚是否额外的细菌因子促进突起的形成和细胞间的传播后，彗星尾形成的步骤。我们的初步数据表明，分泌的李斯特菌毒力蛋白InlC是需要有效的细胞-细胞传播，但不是必不可少的细菌诱导的F-肌动蛋白组装。重要的是，InlC促进宿主ERM蛋白的活化（磷酸化），所述ERM蛋白是已知参与李斯特菌诱导的突起形成和扩散的细胞骨架调节蛋白家族。此外，InlC定位于也含有ERM蛋白ezrin的彗星尾巴的子集。通过酵母双杂交筛选，人衔接蛋白Tuba被鉴定为InlC的配体。像InlC一样，Tuba与Ezrin共同定位在彗星尾巴的一个子集中。Tuba含有几个功能结构域，包括接合ezrin（SH35）或InlC（SH36）的SH3结构域。重要的是，RNA干扰研究表明，Tuba拮抗李斯特菌的细胞间传播。InlC通过减轻Tuba的抑制作用促进扩散，可能是通过损害人SH36配体与Tuba的结合。基于这些结果，似乎InlC、Tuba和ezrin共同作用以促进细胞-细胞扩散。Tuba可能通过影响ERM蛋白或其他效应物来影响传播。 该项目的长期目标是了解InlC促进李斯特菌细胞间传播的分子机制。本提案中的具体目标旨在了解InlC、ERM蛋白、Tuba和Tuba配体如何控制细胞-细胞扩散。 1.确定InlC和/或Tuba是否影响F-肌动蛋白尾部或突起的形成。 2.确定InlC和/或Tuba是否通过ERM蛋白磷酸化控制扩散。 3.评估人类SH36配体在李斯特菌细胞间传播中的作用。 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印迹评价ERM蛋白磷酸化，以及RNA干扰来研究Tuba和Tuba配体在细胞-细胞扩散中的功能。 单核细胞增生李斯特菌是一种食源性细胞内细菌，可导致脑膜炎或流产等严重疾病。李斯特菌通过从一个人体细胞传播到另一个细胞来逃避免疫系统，同时保持在宿主胞质溶胶的保护环境中。这项研究的目的是确定介导李斯特菌传播的细菌和人体成分，这些成分可能适合作为药物靶点。

DESCRIPTION (provided by applicant): Listeria monocytogenes is a food-borne, intracellular pathogen capable of causing gastroenteritis, meningitis, and abortions. 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 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 contact the host cell plasma membrane and induce the formation of Listeria-containing protrusions that are engulfed by neighboring cells. Although ActA-mediated actin polymerization is well understood, it remains unclear whether additional bacterial factors promote protrusion formation and intercellular spread by acting after the step of comet tail formation. Our preliminary data indicate that the secreted Listeria virulence protein InlC is needed for efficient cell-cell spread, but is not essential for bacterial-induced F-actin assembly. Importantly, InlC promotes activation (phosphorylation) of host ERM proteins, a family of cytoskeletal regulatory proteins known to participate in Listeria-induced protrusion formation and spread. Moreover, InlC localizes to a subset of comet tails that also contain the ERM protein ezrin. Through a yeast two-hybrid screen, the human adaptor protein Tuba was identified as a ligand of InlC. Like InlC, Tuba co-localizes with ezrin in a subset of comet tails. Tuba contains several functional domains, including SH3 domains that engage ezrin (SH35) or InlC (SH36). Importantly, RNA interference studies indicate that Tuba antagonizes cell-cell spread of Listeria. InlC promotes spreading by relieving the inhibitory effect of Tuba, possibly by impairing binding of human SH36 ligands to Tuba. Based on these results, it seems likely that InlC, Tuba, and ezrin act together to promote cell-cell spread. Tuba might influence spreading by affecting ERM proteins or other effectors. The long-term goal of this project is to understand the molecular mechanism by which InlC promotes intercellular dissemination of Listeria. The Specific Aims in this proposal are directed towards understanding how InlC, ERM proteins, Tuba, and Tuba ligands control cell-cell spread. 1. Determine if InlC and/or Tuba affect formation of F-actin tails or protrusions. 2. Determine if InlC and/or Tuba control spreading through ERM protein phosphorylation. 3. Evaluate the role of human SH36 ligands in intercellular spread of Listeria. The above objectives will be addressed through a variety of approaches, including immunofluorescence microscopy, evaluation of ERM protein phoshorylation through Western blotting, and RNA interference to investigate the function of Tuba and Tuba ligands in cell-cell spread. Listeria monocytogenes is a food-borne, intracellular bacterium that causes serious illnesses leading to meningitis or abortion. Listeria evades the immune system by spreading from one human cell to another, while remaining within the protective environment of the host cytosol. The goal of this research is to identify bacterial and human components that mediate Listeria spreading, and which may be suitable as drug targets.