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，可引起孕妇、新生儿和免疫功能低下的成年人的严重食源性感染。这种细菌直接在受感染宿主细胞的细胞质中生长，并利用一种基于肌动蛋白的运动形式在受感染细胞内和细胞间迅速移动。一种L. rnonocytogenes蛋白，ActA，诱导宿主细胞肌动蛋白聚合形成“彗星尾”结构，推动细菌穿过宿主细胞质。本研究的总体目标是了解单核增生乳杆菌以肌动蛋白为基础的运动机制。三种互补的方法将被用来研究这个问题：生物化学、生物物理学和细胞生物学。一个主要的目标是建立一个简化的生化系统，可以支持单核增生乳杆菌的运动，并发展定量分析，以便每个成分在肌动蛋白为基础的运动中的精确作用可以评估。移动细菌产生的力的大小将直接使用激光力阱（光学镊子）和柔性微针进行测量。将探讨肌动蛋白丝聚合与运动力产生耦合的机理。最后，视频显微镜技术将用于观察细菌从一个宿主细胞传播到另一个宿主细胞的过程，并结合遗传和药理学扰动来定义细菌、宿主细胞和与移动细菌相关的富含肌动蛋白的彗尾对细胞间传播过程的贡献。成功完成我们的研究目标将对致病菌（如单核增生乳杆菌）与人类宿主细胞特异性交流的机制提供重要的见解。这一认识可能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了解各种基于肌动蛋白的细胞运动的基本生物学过程，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在癌细胞离开肿瘤部位并侵入其他组织之前不会致命，因此，对调节肌动蛋白运动的基本机制的详细了解，可能对开发对抗转移性癌症的治疗策略也很重要。

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.