Regulation of bacterial pathogen actin-based motility by host cell kinases

描述(申请人提供)：几种胞内细菌病原体，如李斯特氏菌、立克次体。和志贺氏菌，依靠基于肌动蛋白的运动从受感染的细胞传播到邻近的细胞，并在宿主内传播。遗传和生化研究表明，基于肌动蛋白的运动依赖于细菌表面毒力因子的产生，这些毒力因子与宿主细胞细胞骨架因子相互作用，包括Arp2/3复合体。尽管有这些开创性的贡献，但支持基于肌动蛋白的运动的宿主细胞因子仍然知之甚少。为了填补这一知识空白，我们开发了结合自动荧光显微镜和计算机辅助图像分析的创新方法，以可视化和量化细菌病原体的传播。我们使用这一新开发的方法来筛选人类基因组，并确定CK1和CK2是李斯特菌传播所需的宿主细胞激酶。我们最近证明，与WASP和WAVE2类似，ActA对Arp2/3复合体的亲和力是由CK2介导的磷酸化调节的，我们称之为调控模仿。在这个提案中，我们提出了遗传和生化方法，旨在探索CK1在调控李斯特菌基于肌动蛋白的运动(Aim1)中的作用(S)。此外，我们建议将用于研究李斯特菌传播的方法扩展到几种显示基于肌动蛋白的运动的细菌病原体，包括志贺氏菌和立克次体(AIM2)。这些探索性研究可能为支持李斯特氏菌、志贺氏菌和立克次体致病的机制以及参与肌动蛋白细胞骨架调控的基本过程提供重要和新颖的见解。 与公共卫生相关：各种细胞内病原体已经进化出操纵宿主细胞过程的能力，以便从受感染的细胞传播到邻近细胞。导致病原体在细胞间传播的大多数宿主因素尚不清楚。在这项提案中，我们提出了我们的计划，以确定和研究参与李斯特氏菌，志贺氏菌的宿主细胞激酶。和立克次体Rickettia spp.基于肌动蛋白的运动性。建议的方法将有助于我们对微生物发病机制的一般理解，因此可能构成合理设计预防和治疗干预措施的基础。

DESCRIPTION (provided by applicant): Several intracellular bacterial pathogens, such as Listeria ssp, Rickettsia spp. and Shigella spp., rely on actin-based motility to spread from infected cells to neighboring cells and disseminate within their host. Genetic and biochemical studies have revealed that actin-based motility relies on the production of virulence factors at the bacterial surface that interact with host cell cytoskeleton factors, including the ARP2/3 complex. In spite of these seminal contributions, the host cell factors supporting actin-based motility are still poorly understood. To fill this gap in knowledge, we have developed innovative approaches combining automated fluorescence microscopy and computer-assisted image analysis in order to visualize and quantify bacterial pathogen spread. We have used this newly developed approach to screen the human kinome and identified CK1 and CK2 as host cell kinases required for Listeria spread. We recently demonstrated that, similar to WASP and WAVE2, the affinity of ActA for the ARP2/3 complex is modulated by CK2-mediated phosphorylation, a notion that we refer to as regulatory mimicry. In this proposal we propose genetic and biochemical approaches designed to explore the role(s) of CK1 in the regulation of Listeria actin-based motility (Aim1). In addition, we propose to extend the approach used for investigating Listeria spread to several bacterial pathogens displaying actin-based motility, including Shigella and Rickettsia (Aim2). These exploratory studies may provide important and novel insights both into the mechanisms supporting Listeria, Shigella and Rickettsia pathogenesis and into the fundamental processes involved in the regulation of the actin cytoskeleton. PUBLIC HEALTH RELEVANCE: Various intracellular pathogens have evolved the ability to manipulate host cell processes in order to spread from infected cells into the neighboring cells. Most of he host factors contributing to the cell-to-cell spread of pathogens are unknown. In this proposal, we present our plans to determine and investigate the host cell kinases involved in Listeria spp., Shigella spp. and Rickettia spp. actin-based motility. The proposed approach will contribute to our general understanding of the mechanisms underlying microbial pathogenesis and may therefore constitute the foundation for the rational design of preventive and therapeutic interventions.