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

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、脑膜炎和流产。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。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。火箭细菌与宿主细胞质膜接触，诱导含有李斯特菌的突起形成，这些突起被邻近细胞吞噬。虽然Acta介导的肌动蛋白聚合已经被很好地理解，但尚不清楚额外的细菌因素是否通过在彗星尾巴形成步骤之后作用而促进突起的形成和细胞间的扩散。我们的初步数据表明，分泌的李斯特菌毒力蛋白InlC是有效的细胞-细胞扩散所必需的，但不是细菌诱导的F-肌动蛋白组装所必需的。重要的是，InlC促进宿主ERM蛋白的激活(磷酸化)，这是一个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，确定人接头蛋白Tuba为InlC的配基。与InlC一样，Tuba与Ezrin共同定位于彗星尾部的一个子集。Tuba包含几个功能结构域，包括与Ezrin(SH35)或InlC(SH36)结合的SH3结构域。重要的是，RNA干扰研究表明，Tuba可以拮抗李斯特菌的细胞间传播。InlC通过减轻Tuba的抑制作用促进传播，可能是通过损害人SH36配体与Tuba的结合。根据这些结果，似乎InlC、Tuba和Ezrin可能共同作用，促进细胞间的扩散。Tuba可能通过影响ERM蛋白或其他效应器来影响传播。 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。这项建议的具体目的是为了了解InlC、ERM蛋白、Tuba和Tuba配体如何控制细胞间的扩散。 1.确定InlC和/或Tuba是否影响F-肌动蛋白尾巴或突起的形成。 2.确定InlC和/或Tuba是否通过ERM蛋白磷酸化控制扩散。 3.评价人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实现，包括免疫荧光显微镜，通过Western blotting评估ERM蛋白的磷酸化，以及RNA干扰来研究Tuba和Tuba配体在细胞-细胞扩散中的功能。 单核细胞增多性李斯特菌是一种食源性细胞内细菌，会导致脑膜炎或流产等严重疾病。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，从而逃避免疫系统。这项研究的目的是确定介导李斯特菌传播的细菌和人类成分，这些成分可能适合作为药物靶标。

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.