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

描述（由申请人提供）：单核细胞增生李斯特菌是一种食源性细胞内病原体，能够引起肠胃炎、脑膜炎和流产。李斯特菌的毒力至关重要的是它在哺乳动物细胞内复制的能力，并通过肌动蛋白依赖的运动过程从一个细胞传播到另一个细胞，这使得细菌能够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发起的，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。火箭细菌接触宿主细胞质膜并诱导形成含有李斯特菌的突起，这些突起被邻近细胞吞没。虽然acta介导的肌动蛋白聚合已被很好地理解，但尚不清楚是否有其他细菌因子在彗星尾形成后作用，促进了突起的形成和细胞间扩散。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-细胞传播所必需的，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），ERM蛋白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定位于含有ERM蛋白ezrin的彗尾子集。通过酵母双杂交筛选，鉴定了人接头蛋白Tuba为InlC的配体。像InlC一样，Tuba与ezrin在彗星尾巴的一个子集中共同定位。Tuba包含几个功能域，包括与ezrin （SH35）或InlC （SH36）相关的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-细胞传播。InlC通过减轻Tuba的抑制作用来促进扩散，可能是通过损害人类SH36配体与Tuba的结合。基于这些结果，InlC、Tuba和ezrin似乎可能共同作用，促进细胞间的扩散。大号可能通过影响ERM蛋白或其他效应器来影响扩散。

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.