ACTIN BASED MOTILITY OF A BACTERIAL PATHOGEN

李斯特菌是一种普遍存在的革兰氏阳性细菌，可导致孕妇、新生儿和免疫功能低下的成年人发生严重的食源性感染。细菌直接生长在受感染宿主细胞的细胞质中，并利用一种基于肌动蛋白的运动形式在感染细胞内和感染细胞之间快速移动。一种嗜酸乳杆菌蛋白ActA能诱导宿主细胞肌动蛋白聚合，形成“彗星尾巴”结构，推动细菌穿过宿主细胞细胞质。这项建议的总体目标是了解基于肌动蛋白的单核细胞增多性李斯特菌的运动机制。将使用三种互补的方法来研究这个问题：生化、生物物理和细胞生物学。一个主要的目标是建立一种能够支持单核细胞增多性李斯特氏菌运动的简化生化系统，并开发定量分析方法，以便能够评估每种成分在基于肌动蛋白的运动中的准确作用。移动细菌产生的力的大小将使用激光测力器(光镊子)和顺应性微针直接测量。我们将研究肌动蛋白细丝聚合和运动力产生之间的耦合机制。最后，将使用视频显微镜技术观察细菌从一个宿主细胞传播到另一个宿主细胞的过程，并将使用遗传和药理学扰动的组合来确定细菌、宿主细胞和与移动的细菌相关的富含肌动蛋白的彗星尾巴对细胞间传播过程的贡献。成功完成我们的研究目标将使我们对单核细胞增多性乳杆菌等病原菌与其人类宿主细胞进行特定通信的机制有重要的了解。这一认识可能会为开发预防和治疗细菌感染的新方法铺平道路。此外，我们的研究结果将有助于我们理解涉及基于肌动蛋白的细胞运动的各种基本生物学过程，包括伤口愈合、免疫系统反应和胚胎发育。此外，由于大多数恶性肿瘤直到癌细胞离开肿瘤部位并侵入其他组织时才会变得致命，因此详细了解调节基于肌动蛋白的运动的基本机制对于制定抗击转移癌的治疗策略也可能是重要的。

Listeria rnonocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised adults. The bacterium grows directly in the cytoplasm of infected host cells and moves rapidly throughout and between infected cells using a form of actin-based motility. An L. rnonocytogenes protein, ActA, induces polymerization of host cell actin to form a "comet tail" structure that pushes the bacterium through the host cell cytoplasm. The overall goal of this proposal is to understand the mechanism of the actin-based motility of L. monocytogenes. Three complementary approaches will be used to study this problem: biochemical, biophysical, and cell biological. A major goal is the establishment of a simplified biochemical system that can support L. monocytogenes motility, and development of quantitative assays so that the precise role of each component in actin-based motility can be assessed. The amount of force generated by moving bacteria will be measured directly using a laser force trap (optical tweezers) and compliant microneedles. The mechanism of coupling between actin filament polymerization and production of a motile force will be examined. Finally, videomicroscopy techniques will be used to observe the process of bacterial spread from one host cell to another, and a combination of genetic and pharmacological perturbations will be used to define the contributions of the bacterium, the host cell, and the actin-rich comet tail associated with the moving bacterium, to the process of intercellular spread. Successful completion of our research goals would give significant insight into the mechanisms by which pathogenic bacteria such as L. monocytogenes communicate specifically with the cells of their human hosts. This understanding might pave the way for the development of new ways to prevent and cure bacterial infections. In addition, the results of our research would contribute to our understanding of a wide variety of basic biological process involving actin-based cell movement, including wound healing, immune system responses, and embryonic development. Furthermore, since most malignant tumors do not become lethal until the cancer cells move away from the tumor site and invade other tissues, a detailed understanding of the basic mechanisms that regulate actin-based motility may also be important in the development of therapeutic strategies for combating metastatic cancers.