Actin Based Motility of a Bacterial Pathogen

描述(申请人提供)：单核细胞增生性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受到严重的食源性感染。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。单核细胞增多性李斯特菌表面蛋白ActA以极化方式表达，并与宿主细胞细胞骨架因子相互作用，诱导肌动蛋白“彗星尾”结构聚合，推动细菌穿过宿主细胞细胞质。本项目的总体目标是了解单核细胞增多性李斯特氏菌基于肌动蛋白的运动的机制和生物学意义。我们结合使用三种互补的方法来研究这种形式的运动性--生物物理、生化和细胞生物学。单核细胞增多性李斯特氏菌基于肌动蛋白的运动所需的一整套基本蛋白质组分现在已经被确定，该领域开始就理解稳态运动中的力产生的一般物理框架达成一致。接下来的一系列概念和实验挑战在于重建和掌握生物行为全部复杂性背后的机制。我们将在现有的稳态模型不能充分解决的两个领域中，重点研究基于肌动蛋白的细菌运动的机制：了解细菌运动启动的生化和生物物理机制，以及探索负责调节路径持续性和曲率的决定因素。在细菌细胞间传播的背景下研究感染的细胞生物学时，我们将开发新的技术来直接观察组织培养中完全极化的上皮单层中的细菌传播以及上皮细胞和巨噬细胞之间的异型传播。此外，我们将在确定ActA蛋白极化机制的过程中解决细菌细胞的细胞生物学问题。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。此外，由于单核细胞增生性李斯特氏菌基于肌动蛋白的运动是一个通过肌动蛋白聚合产生力量的简单模型系统，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、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 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. 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 use a combination of three complementary approaches to studying this form of motility-biophysical, biochemical, and cell biological. The full set of basic protein components required for actin-based motility by L. monocytogenes have now been identified and the field is beginning to agree on a general physical framework for understanding force generation during steady-state movement. The next set of conceptual and experimental challenges lie in rebuilding and coming to grips with the mechanisms underlying the full complexity of the biological behavior. We will focus our mechanistic studies on bacterial actin-based motility in two areas that are not adequately addressed by the existing steady-state models: understanding the biochemical and biophysical mechanism of bacterial movement initiation and exploring the determinants responsible for regulating path persistence and curvature. In examining the cell biology of infection in the context of bacterial cell-to-cell spread, we will develop new techniques for direct observation of bacterial spread in fully polarized epithelial monolayers in tissue culture and heterotypic spread between epithelial cells and macrophages. In addition, we will address the cell biology of the bacterial cell in determining the mechanism of ActA protein polarization. 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. In addition, since 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.