Structural Studies of Bacterial Virulence Factors

描述(申请人提供)：许多动植物病原体利用一种类似的分泌系统，称为病毒型或“接触依赖型”，将一组细菌效应蛋白输送到宿主细胞中。鼠伤寒沙门氏菌使用这种分泌系统来注射操纵宿主细胞功能的蛋白质，以诱导细菌进入肠道上皮细胞中正常的非吞噬细胞。这一过程依赖于不到10个易位的效应器蛋白，这些蛋白协同诱导戏剧性的膜褶皱，导致细菌通过巨噬细胞吞噬而内化。 这项工作的长期目标是使用结构生物学作为分子理解这种病原体入侵过程的基础，并利用这些信息来识别潜在的药物筛选靶点。这项建议的具体目的是(1)确定与鼠伤寒沙门氏菌入侵相关的易位效应因子的结构，(2)确定这些因子与其宿主细胞靶标的共晶结构，以及(3)在细菌宿主细胞入侵和细胞骨架操纵的背景下，使用基于结构的突变来检查这些因子的相互作用表面。因此，这项工作将涉及一种结合大分子X射线结晶学、生化分析和微生物细胞生物学的多学科方法。 细菌感染现在和过去都是造成死亡和人类痛苦的重要原因。不祥的是，我们对抗细菌病原体的武器现在正在失效，因为越来越多的微生物对更多的药物产生了抗药性。此外，使用微生物制剂作为战争或恐怖主义工具的威胁越来越大，这已成为一个非常现实的关切。因此，这些研究的最终目的将是利用结构信息来帮助选择靶标，以筛选将损害这种病原体的毒力机制的抑制化合物，并作为开发针对其他感染性细菌生物体的类似策略的范例。

DESCRIPTION (provided by applicant): Many animal and plant pathogenic bacteria utilize a similar secretion system, termed type Ill or "contact dependent," to deliver a battery of bacterial effector proteins into host cells. Salmonella typhimurium uses such a secretion system to inject proteins that manipulate host cellular functions to induce the uptake of the bacterium into the normally non-phagocytic cells of the intestinal epithelium. This process relies on less than ten translocated effectors proteins, which collaborate to induce dramatic membrane ruffling, leading to bacterial internalization by macropinocytosis. The long-term goal of this work is to use structural biology as a foundation for a molecular understanding of the invasion process of this pathogen, and to exploit this information in the identification of potential targets for drug screening. The specific aims of this proposal are (1) to determine structures of S. typhimurium invasion-associated translocated effectors, (2) to determine the co-crystal structures of these factors with their host cell targets, and, finally, (3) to use structure-based mutagenesis to examine the interacting surfaces of these factors in the context of bacterial host cell invasion and cytoskeletal manipulation. This work will thus involve a multidisciplinary approach combining macromolecular X-ray crystallography, biochemical assays, and microbial cell biology. Bacterial infection is and has been a significant cause of death and human suffering. Ominously, our weapons for combating bacterial pathogens are now failing as ever-increasing numbers of microorganisms have developed resistance to greater numbers of our drugs. Furthermore, the increased threat of the use of microbial agents as instruments of war or terrorism has become a very real concern. Therefore, a final aim of these studies will be to use the structural information to aid in selecting targets for the screening of inhibitory compounds that will impair the virulence mechanisms of this pathogen, and to serve as a paradigm for developing similar strategies against other infectious bacterial organisms.