LISTERIA AND SHIGELLA USE ACTIN TO SPREAD CELL TO CELL

描述（由申请人提供）：革兰氏阳性杆菌单核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氏杆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，称为丝状足。丝状足进入邻近的细胞，在那里它们被摄入，允许有效的细胞间扩散和避免体液免疫系统，以及许多抗生素。肌动蛋白调控蛋白和介导细菌诱导丝状足形成的途径尚不清楚。我们将：目的一-探索已知的肌动蛋白调节蛋白对李斯特菌和志贺氏菌诱导的丝状足形成的相对贡献。免疫荧光和免疫金电镜、GFP融合蛋白、RNAi和敲除细胞系的表达将结合冷冻断裂电镜、光漂后荧光恢复（FRAP）和激光纳米剪刀来评估肌动蛋白调控蛋白Eps8、VASP、Arp2/3复合物、束状蛋白和肌球蛋白x在丝状足形成中的作用。目的II -剖析启动李斯特菌诱导的丝状足形成的信号转导途径。PI3K抑制剂LY294002阻断李斯特菌诱导的丝状伪足形成，PIP3的传递导致丝状伪足数量增加bb60倍，而李斯特菌细胞内运动没有明显变化。使用靶向RNAi敲除，将确定启动丝状足形成的特定PI3K同型。下游影响因子Akt、PKC-gamma、ttap -1和Plekstrin-2、GAPs、gef和小g蛋白的作用将被检测。通过Fura-2和BAPTAM评估了丝状足形成对[Ca2+]i变化的响应，我们发现Ca2+螯合后丝状足长度增加了100倍。将探讨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。这些研究有望阐明李斯特菌和志贺氏菌诱导的丝状足形成和细胞间传播所需的关键调控途径、毒力的关键步骤和新的治疗方法。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。

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.