Structural Studies of Bacterial Virulence Factors

描述（由申请人提供）：许多动物和植物病原性细菌利用类似的分泌系统，称为III型或“接触依赖性”，以将一系列细菌效应蛋白递送到宿主细胞中。鼠伤寒沙门氏菌使用这样的分泌系统来注射操纵宿主细胞功能的蛋白质，以诱导细菌摄取到肠上皮的正常非吞噬细胞中。这一过程依赖于不到10个易位效应蛋白，它们共同诱导戏剧性的膜皱褶，导致细菌通过巨胞饮内化。 这项工作的长期目标是利用结构生物学作为基础，从分子上了解这种病原体的入侵过程，并利用这些信息来确定药物筛选的潜在靶点。本研究的具体目的是：（1）确定S.鼠伤寒杆菌侵袭相关的易位效应物，（2）确定这些因子与其宿主细胞靶的共晶体结构，以及，最后，（3）使用基于结构的诱变来检查这些因子在细菌宿主细胞侵袭和细胞骨架操纵的背景下的相互作用表面。因此，这项工作将涉及一个多学科的方法相结合的大分子X射线晶体学，生化分析，和微生物细胞生物学。 细菌感染是并且一直是死亡和人类痛苦的重要原因。不幸的是，我们对付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我们越来越多的药物产生了抗药性。此外，使用微生物剂作为战争或恐怖主义工具的威胁日益增加，已成为一个非常真实的关切。因此，这些研究的最终目的将是使用结构信息来帮助选择用于筛选抑制性化合物的靶标，所述抑制性化合物将损害该病原体的毒力机制，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.