Actin-Based Motility of a Bacterial Pathogen

描述（由申请人提供）：单核增生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、免疫功能低下或老年人严重的食源性感染。由于单核细胞增生乳杆菌具有突破血脑屏障的特殊能力，一些患者出现中枢神经系统感染。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。单核增生乳杆菌表面蛋白ActA以极化方式表达，并与宿主细胞骨架因子相互作用，诱导肌动蛋白“彗星尾”结构聚合，推动细菌通过宿主细胞质。本项目的总体目标是了解单核增生乳杆菌肌动蛋白运动的机制和生物学意义。我们建议采用跨学科的方法在各个层面上研究这种运动形式，从参与肌动蛋白运动产生的单分子的生物化学和生物物理学到感染动物血脑屏障传播的动力学。在分子水平上，我们将确定肌动蛋白彗尾组装的最小功能单元。在细菌细胞水平，我们将建立细菌细胞壁合成和重塑如何决定ActA定位，并测量ActA错定位对肌动蛋白彗尾形成和运动的影响。在宿主细胞水平上，我们将鉴定宿主细胞中对细胞间扩散过程有特殊贡献的成分，并阐明细菌穿越内皮单层的可能机制。在宿主动物的水平上，我们将在新开发的全身感染小鼠模型中确定细菌如何穿过血脑屏障。我们的研究目标的成功完成将对致病细菌（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，从而破坏宿主细胞功能，有利于细菌的繁殖和传播。此外，由于单核增生乳杆菌基于肌动蛋白的运动是肌动蛋白聚合产生力的一个简单模型系统，我们的研究结果将有助于我们了解与肌动蛋白为基础的细胞运动有关的各种基本生物学过程，包括伤口愈合、炎症、胚胎发育和癌症转移。

DESCRIPTION (provided by applicant): Listeria monocytogenes is a ubiquitous Gram-positive bacterium that can cause serious food-borne infections in pregnant women, newborns and immunocompromised or older adults. Some patients develop infections in the central nervous system because of the unusual ability of L. monocytogenes to breach the blood-brain barrier. 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. The L. monocytogenes surface protein, ActA, is expressed in a polarized fashion and interacts with host cell cytoskeletal factors to induce the polymerization of an actin "comet tail" structure that pushes the bacterium through the host cell cytoplasm. The overall goal of this project is to understand the mechanism and biological significance of the actin-based motility of L. monocytogenes. We propose to use an interdisciplinary approach to studying this form of motility at all levels from the biochemistry and biophysics of single molecules involved in the generation of actin-based motility to the dynamics of spread across the blood-brain barrier in infected animals. At the MOLECULAR level, we will determine the minimal functional unit for actin comet tail assembly. At the level of the BACTERIAL CELL, we will establish how ActA localization is determined by bacterial cell wall synthesis and remodeling, and measure the effects of ActA mislocalization on actin comet tail formation and motility. At the level of the HOST CELL, we will identify host cell components that contribute specifically to the process of cell- to-cell spread, and elucidate the possible mechanisms by which bacteria can cross endothelial monolayers. At the level of the HOST ANIMAL, we will determine how the bacteria cross the blood-brain barrier in a newly developed mouse model of systemic infection. 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 to subvert host cell function in favor of bacterial propagation and dissemination. In addition, because L. monocytogenes actin-based motility is a simple model system for force generation by actin polymerization, the results of our research would contribute to our understanding of a wide variety of basic biological processes involving actin-based cell movement, including wound healing, inflammation, embryonic development, and cancer metastasis. PUBLIC HEALTH RELEVANCE: Listeria monocytogenes bacteria cause a rare but very serious form of food poisoning, which can lead to meningitis (infection of the brain and central nervous system) in newborns, immunocompromised people, and older adults, and can also cause late-term miscarriage in pregnant women. These bacteria grow directly inside of human host cells and use their niche inside of human cells to "hide out" from antibodies that the immune system normally generates to fight infections. The short-term goal of this research is to understand how Listeria monocytogenes are able to invade human cells and move from the inside of one human cell to another, with a long-term goal of finding better methods to prevent or treat serious bacterial infections.