LISTERIA AND SHIGELLA USE HOST CELL ACTIN

描述（由申请人提供）：革兰氏阳性杆菌单核增生李斯特菌主要感染免疫功能低下患者，引起菌血症和脑膜炎，而革兰氏阴性杆菌福氏志贺氏杆菌感染正常宿主，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。李斯特菌和志贺氏菌诱导宿主细胞肌动蛋白组装成火箭尾巴，迅速推动细菌通过细胞质，允许它们在细胞间传播，并避免体液免疫系统。肌动蛋白的组装发生在一个离散的聚合区，就在活动细菌的后面。这个区域阻断宿主细胞肌动蛋白调节蛋白，凝胶蛋白，CapZ和CapG，这些蛋白通常覆盖在肌动蛋白丝的快速生长末端。这种阻断活性允许肌动蛋白丝在这个离散区快速组装。其中两种蛋白质，凝胶蛋白和CapG，需要微摩尔钙才能发挥作用。我们将：目的一-阐明李斯特菌如何在聚合区阻断倒钩端盖蛋白。Pyrenyl肌动蛋白和直角光散射将用于研究profilin联合PIP2和VASP或N-WASP如何影响CapG， CapZ和gelsolin对肌动蛋白丝的覆盖。李斯特菌的封盖抑制作用将在profilin和VASP耗尽前后的无脑细胞提取物中进行研究。在李斯特菌和志贺氏菌感染的细胞中，PIP2（众所周知的阻断帽盖活性）的定位将使用GFP标记探针进行研究。使用PI激酶抑制剂Wortmannin和槲皮素阻断PIP2产生的效果，感染缺乏PIP2结合位点的李斯特菌ActA突变体和缺乏VASP结合位点的ActA突变体的细胞，将进行研究。目的二：研究李斯特菌和志贺氏菌肌动蛋白运动对钙的依赖性。钙是开启和关闭肌动蛋白调控蛋白的关键信号，我们发现螯合剂BAPTAM阻断志贺氏菌肌动蛋白的运动，减缓李斯特菌火箭尾的分解。将研究N-WASP和vinculin（志贺氏菌诱导的肌动蛋白组装所特有的细胞蛋白）以及gelsolin的Ca2+敏感性。这些研究应阐明李斯特菌和志贺氏菌诱导的肌动蛋白组装所需的关键调控途径，并可能确定治疗李斯特菌病和志贺氏菌病的新治疗靶点。

DESCRIPTION (provided by applicant): The gram-positive bacillus Listeria monocytogenes predominantly infects immunocompromised patients, causing bacteremia and meningitis while the gram-negative bacillus Shigella flexneri infects normal hosts causing severe diarrhea and dehydration. The pathogenesis of Listeriosis and Shigellosis absolutely requires these intracellular bacteria to usurp the host cell's contractile system. Listeria and Shigella induce host cell actin to assemble into rocket tails that rapidly propel the bacteria through the cytoplasm, allowing their cell-to-cell spread and avoidance of the humoral immune system. Actin assembly occurs in a discrete polymerization zone directly behind the motile bacteria. This region blocks the host cell actin-regulatory proteins, gelsolin, CapZ and CapG, that normally cap the fast growing ends of actin filaments. This blocking activity allows actin filaments to rapidly assemble in this discrete zone. Two of these proteins, gelsolin and CapG, require micromolar calcium to function. We will: Aim I - Elucidate how Listeria blocks barbed end-capping proteins in the polymerization zone. Pyrenyl actin and right angle light scattering will be used to examine how profilin combined PIP2 and VASP or N-WASP effects actin filament capping by CapG, CapZ and gelsolin. Capping inhibition by Listeria will be investigated in brain cell free extracts before and after depletion of profilin and VASP. Localization of PIP2 (well known to block capping activity) in Listeria and Shigella infected cells will be studied using a GFP labeled probe. The effects of blocking PIP2 production using the PI kinase inhibitors Wortmannin and quercetin, infecting cells with Listeria ActA mutants lacking PIP2 binding sites, and ActA mutants lacking VASP binding sites will be examined. Aim II - Study the Calcium-Dependence of Listeria and Shigella actin-based motility. Calcium is a critical signal for turning on and off actin regulatory proteins, and we have found that the chelator BAPTAM blocks Shigella actin-based motility and slows the disassembly of Listeria rocket tails. The Ca2+-sensitivity of N-WASP and vinculin, cell proteins unique to Shigella-induced actin assembly, as well as gelsolin will be studied. These investigations should clarify key regulatory pathways required for Listeria- and Shigella-induced actin assembly and may identify new therapeutic targets for treating Listeriosis and Shigellosis.