Host actin cytoskeleton rearrangements induced by Salmonella

描述(由申请人提供)：入侵非吞噬宿主细胞的能力是沙门氏菌毒力的关键。SipA、SIPC、SopB、SopE和SopE2是五种已知的通过直接和间接调节宿主肌动蛋白动态来促进沙门氏菌侵袭的III型效应器。SIPA和SIPC通过模拟宿主肌动蛋白结合蛋白的功能直接调节肌动蛋白的动态。SopB、SopE和SopE2间接地颠覆宿主细胞信号转导通路，诱导肌动蛋白细胞骨架重排和膜褶皱。这些细菌效应蛋白的协同作用最终导致细菌的有效吸收。SIPA、SIPC、SopB是多种功能的效应物，也参与了效应器的移位和诱导宿主的炎症反应。尽管对这些效应因子及其在沙门氏菌诱导的肌动蛋白细胞骨架重排中的单独作用有了更深入的了解，但很少有人将它们的多功能活性分离出来，也没有研究这些活性是如何在功能上相互作用以促进沙门氏菌的侵袭。在这项研究中，我们建议调查它们对肌动蛋白调节活性的个体贡献，以及它们在细菌入侵过程中如何在功能上进行协调。我们的初步数据显示，SIPC通过瞬时二聚体的形成发挥其肌动蛋白成核活性，SIPA和SIPC可能在功能上协同促进细菌进入。我们还证明了SopB依赖的VAMP8重新募集到沙门氏菌诱导的膜皱纹，并且VAMP8是有效的细菌入侵所必需的。我们进一步确定SIPA和SIPC可能介导SopB和Sope非依赖性沙门氏菌进入。我们推测SipA、SIPC、SopB和Sope在调节宿主肌动蛋白动态以促进沙门氏菌进入的过程中既有协同作用又有独立作用。为了验证这一假设，我们将：(I)表征SipA、SIPC肌动蛋白调控活性在沙门氏菌入侵中的作用；(Ii)确定SopB促进肌动蛋白细胞骨架重排和沙门氏菌入侵的分子机制；(Iii)研究SipA、SIPC、SopB和SOPE如何协调其肌动蛋白调控活性促进沙门氏菌入侵。本研究的结果将为SipA、SIPC和SopB如何独立和协同地利用宿主肌动蛋白细胞骨架促进沙门氏菌进入提供分子基础。 项目简介：尽管我们对沙门氏菌研究的长期兴趣获得了理解，但沙门氏菌病继续构成世界范围内的医学问题，即使在发达国家，沙门氏菌病仍然是食源性疾病的头号原因。沙门氏菌毒力蛋白宿主细胞靶点的确定将有助于临床治疗药物的设计和多重耐药细菌的治疗。这项研究的结果不仅将拓宽我们对沙门氏菌发病机制的理解，还将为基础宿主细胞生物学提供重要的见解。

DESCRIPTION (provided by applicant): The ability to invade non-phagocytic host cells is essential for Salmonella virulence. SipA, SipC, SopB, SopE, and SopE2 are five type III effectors that are known to promote Salmonella invasion by modulating host actin dynamics both directly and indirectly. SipA and SipC regulate actin dynamics directly by mimicking functions of host actin-binding proteins. Indirectly, SopB, SopE, and SopE2 subvert host cell signal transduction pathways to induce actin cytoskeletal rearrangements and membrane ruffling. The concerted actions of these bacterial effector proteins eventually lead to the efficient uptake of the bacterium. SipA, SipC, SopB are multifunctional effectors also involved in effector translocation and in inducing host inflammatory responses. Despite improved understanding of these effectors and their individual roles in Salmonella-induced actin cytoskeleton rearrangements, little has been done to separate their multifunctional activities and to examine how these activities functionally interact to promote Salmonella invasion. In this study, we propose to investigate the individual contributions of their actin-modulating activities and how they are functionally coordinated during bacterial invasion. Our preliminary data showed that SipC exerts its actin-nucleation activity through transient dimer formation and that SipA and SipC may functionally cooperate to promote bacterial entry. We also demonstrated SopB-dependent VAMP8 recruitment to Salmonella-induced membrane ruffles and that VAMP8 is required for efficient bacterial invasion. We further determined that SipA and SipC may mediate SopB- and SopE-independent Salmonella entry. We hypothesize that SipA, SipC, SopB, and SopE function both cooperatively and independently to regulate host actin dynamics to promote Salmonella entry. To test this hypothesis, we will: (i) characterize the contributions of SipA, SipC actin-modulating activities in Salmonella invasion; (ii) define the molecular mechanism by which SopB promotes actin cytoskeleton rearrangements and Salmonella invasion; (iii) study how SipA, SipC, SopB, and SopE coordinate their actin-modulating activities to promote Salmonella invasion. Results from this study will provide a molecular basis for how SipA, SipC and SopB exploit the host actin cytoskeleton both independently and cooperatively to promote Salmonella entry. Project Narrative: Despite the understanding gained by our long-standing interest in studying Salmonella, salmonellosis continues to pose worldwide medical concerns and remains the number one cause of food-borne diseases even in developed countries. The identification of host cellular targets of Salmonella virulence proteins will aid clinical therapeutic drug designs and treatment of multidrug-resistant bacteria. Results from this study will not only broaden our understanding of Salmonella pathogenesis but will also provide vital insights into basic host cell biology.