Bifunctional Control of Yop Translocation by YopK

描述（由申请人提供）：III型分泌是一种通过分子注射器样结构将毒性蛋白直接递送到宿主细胞中的方法，其被许多革兰氏阴性病原体采用。III型分泌系统导致人类多种疾病，包括腺鼠疫、食源性疾病和囊性纤维化。仅每年数百万例食源性疾病就对我们的卫生保健系统产生了惊人的影响。再加上新出现的抗生素耐药性越来越致命的潜力，迫切需要新的抗菌治疗。虽然由单个病原体递送的毒素不同，但组装和部署III型分泌系统的基本原理是很好地保守的，并且代表了潜在的治疗靶点。为了设计有效的策略，使这种毒力因子失效， 监管是必要的。 III型分泌系统通常仅在易感宿主感染期间产生和激活。在感染过程中，细菌组装针状结构，但毒素不会通过它分泌，直到与宿主细胞接触。细菌如何在细胞接触时激活分泌系统？同样重要的是，他们是如何关闭的？这些问题对于我们理解该系统至关重要，也是本提案的重点。 我们研究耶尔森氏菌III型分泌系统，因为它是一个完善的模型系统。我们的新工具和方法揭示了对III型分泌调节心脏的新见解。特别是，我们发现YopK，一种III型分泌的调节剂，具有两种不同的调节功能，在“针”的两端都起作用。看来YopK是分泌的ON/OFF开关的组成部分。从细菌方面来说，YopK有助于监测哪些蛋白质通过针头分泌。YopK本身是分泌的，一旦进入宿主细胞，YopK与针复合物的末端相互作用以关闭毒素的分泌。在本提案中，我们将研究YopK如何执行这些活动。使用遗传学，细胞生物学和生物化学的组合，我们将确定YopK如何与针两侧的其他已知分泌调节剂协调。我们还将确定和表征YopK内介导其活动的功能结构域。总的来说，我们的工作将揭示控制这一重要毒力武器的基本机制。如果我们理解了ON/OFF开关的本质，我们就可以设计一种新型的广谱药物，靶向广泛细菌的重要分泌系统。

DESCRIPTION (provided by applicant): Type III secretion is a method of delivering toxic proteins directly into host cells via a molecular syringe- like structure, which is employed by many Gram-negative pathogens. Type III secretion systems contribute to an incredibly diverse range of diseases in humans including bubonic plague, food-borne illnesses, and cystic fibrosis. The millions of cases each year of food-borne illnesses alone have a staggering impact on our health care system. Add to that the increasingly deadly potential from emerging antibiotic resistance, and the need for new antimicrobial treatments is urgent. Though the toxins that are delivered by individual pathogens vary, the fundamentals of assembling and deploying type III secretion systems are well conserved and represent a potential therapeutic target. To design effective strategies that disable this virulence factor, a better understanding of its architecture and regulation is necessary. Type III secretion systems are generally only made and activated during infection of a susceptible host. During infection, bacteria assemble the needle-like structure, but toxins are not secreted through it until contact with a host cell is made. How do bacteria activate the secretion system upon cell contact? Just as important, how do they shut if off? These questions are fundamental to our understanding of the system, and these questions are the focus of this proposal. We study the Yersinia type III secretion system because it is a well-established model system. Our novel tools and approaches have revealed new insight into the heart of type III secretion regulation. In particular, we have found that YopK, a regulator of type III secretion, has two distinct regulatory functions and works at both ends of the "needle". I appears that YopK is an integral component of an ON/OFF switch for secretion. From the bacterial side, YopK helps to monitor which proteins are secreted through the needle. YopK itself is secreted and once inside the host cell, YopK interacts with the end of the needle complex to shut down secretion of the toxins. In this proposal we will investigate how YopK performs these activities. Using a combination of genetics, cell biology, and biochemistry we will determine how YopK coordinates with other known secretion regulators on both sides of the needle. We will also identify and characterize the functional domains within YopK that mediate its activities. Collectively, our work will reveal fundamental mechanisms controlling this important virulence weapon. If we understand the nature of the ON/OFF switch, we can design a new type of broad-spectrum drug that targets a vital secretion system in a wide range of bacteria.