LISTERIA AND SHIGELLA USE ACTIN TO SPREAD CELL TO CELL

描述（由申请方提供）：革兰氏阳性杆菌单核细胞增生李斯特菌引起菌血症和脑膜炎，而革兰氏阴性杆菌福氏志贺菌引起严重腹泻和脱水。李斯特菌病和志贺氏菌病的发病机制绝对需要这些细胞内细菌篡夺宿主细胞的收缩系统，形成膜突起，称为丝状伪足。丝状伪足推进到邻近的细胞中，在那里它们被摄入，允许有效的细胞间传播，避免体液免疫系统以及许多抗生素。肌动蛋白调节蛋白和介导细菌诱导的丝状伪足形成的途径知之甚少。我们将：目的I -探索已知的肌动蛋白调节蛋白的相对贡献李斯特菌和志贺氏菌诱导丝状伪足的形成。免疫荧光和免疫金电子显微镜、GFP融合蛋白表达、RNAi和敲除细胞系将与冷冻断裂电子显微镜、光漂白后荧光恢复（FRAP）和激光纳米剪刀相结合，以评估肌动蛋白调节蛋白Eps 8、VASP、Arp 2/3复合物、肌成束蛋白和肌球蛋白X在丝状伪足形成中的作用。目的II -解剖启动李斯特菌诱导的丝状伪足形成的信号转导途径。PI 3 K抑制剂LY 294002阻断李斯特菌诱导的丝状伪足形成，并且PIP 3的递送导致丝状伪足数量增加> 6倍，而李斯特菌细胞内运动性没有显著变化。使用靶向RNAi敲低，将确定负责起始丝状伪足形成的特定PI 3 K同种型。将检查下游影响因子Akt、PKC-γ、Tapp-1和Plekstrin-2、GAP、GEF和小G蛋白的作用。Fura-2和BAPTAM正在评估响应于[Ca 2 +]i变化的丝状伪足形成，并且我们发现在Ca 2+螯合后丝状伪足长度增加> 2倍。多胺钙通道激动剂和拮抗剂对李斯特菌细胞间传播的影响将被探索。这些研究有望澄清李斯特菌和志贺氏菌诱导的丝状伪足形成和细胞间传播所需的关键调控途径，毒力和新的治疗方法的关键步骤。这些发现可以应用于所有形式的细胞运动，包括肿瘤转移，神经突生长和血小板扩散。 公共卫生相关性：李斯特菌是致命的食物中毒和志贺氏菌可导致严重的出血性腹泻。我们正在研究这些侵略性细菌通过秘密地在我们的细胞内移动并避开抗生素来接管我们身体的方式。了解这些微生物如何接管我们细胞的肌肉系统可能会导致对这些危险感染的新治疗方法，并提供对癌细胞如何在我们体内扩散，神经细胞生长和血小板控制出血的新理解。

DESCRIPTION (provided by applicant): The gram positive bacillus Listeria monocytogenes causes bacteremia and meningitis, while the gram negative bacillus Shigella flexneri causes severe diarrhea and dehydration. The pathogenesis of Listeriosis and Shigellosis absolutely requires these intracellular bacteria to usurp the host cell's contractile system to form membrane projections, called filopodia. Filopodia push into adjacent cells where they are ingested, allowing efficient cell-to-cell to spread and avoidance of the humoral immune system, as well as many antibiotics. The actin regulatory proteins and pathways that mediate bacteria-induced filopodia formation are poorly understood. We will: Aim I - Explore the relative contribution of known actin-regulatory proteins to Listeria- and Shigella-induced filopodia formation. Immuno-fluorescence and immuno-gold electron microscopy, expression of GFP fusion proteins, RNAi, and knockout cell lines will be combined with freeze fracture electron microscopy, fluorescence recovery after photo-bleaching (FRAP), and laser nano-scissors to assess the roles of the actin-regulatory proteins Eps8, VASP , Arp2/3 complex, fascin, and myosin-X in filopodia formation. Aim II - Dissect the signal transduction pathways that initiate Listeria-induced filopodia formation. The PI3K inhibitor LY294002 blocks Listeria-induced filopodia formation, and delivery of PIP3 results in a > 6 x increase in filopodia number without a significant change in Listeria intracellular motility. Using targeted RNAi knockdown, the specific PI3K isotype responsible for initiating filopodia formation will be determined. The role of the downstream affecters Akt, PKC-gamma, Tapp-1 and Plekstrin-2, GAPs, GEFs, and small G-proteins will be examined. Filopodia formation in response to changes in [Ca2+]i is being assessed by Fura-2 and BAPTAM, and we find a > 2x increase in filopodia length following Ca2+ chelation. The effects of polyamine calcium channel agonist and antagonists on Listeria cell-to-cell spread will be explored. These investigations promise to clarify the key regulatory pathways required for Listeria- and Shigella-induced filopodia formation and cell-to-cell spread, critical steps for virulence and new therapeutic approaches. These discoveries can be applied to all forms of cell motility, including tumor metastasis, neurite outgrowth, and platelet spreading. PUBLIC HEALTH RELEVANCE: Listeria is responsible for deadly food poisoning and Shigella can cause severe bloody diarrhea. We are examining the ways these aggressive bacteria take over our bodies by secretly moving within our cells and avoiding antibiotics. Understanding how these microbes take over our cells' muscle systems may lead to new treatments for these dangerous infections, as well as provide a new understanding of how cancer cells spread in our body, nerve cells grow, and platelets control bleeding.