Bifunctional Control of Yop Translocation by YopK

描述(申请人提供)：III型分泌物是一种通过分子注射器状结构将有毒蛋白质直接输送到宿主细胞的方法，许多革兰氏阴性病原体都使用这种结构。III型分泌系统导致人类一系列令人难以置信的疾病，包括腺鼠疫、食源性疾病和囊性纤维化。仅每年数以百万计的食源性疾病病例就对我们的医疗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带来的日益致命的潜力，迫切需要新的抗菌治疗方法。虽然不同病原体传递的毒素不同，但组装和部署III型分泌系统的基本原理是保守的，是一个潜在的治疗靶点。为了设计有效的策略来禁用这种毒力因素，更好地理解它的架构 监管是必要的。III型分泌系统一般只有在感染敏感宿主时才会产生和激活。在感染期间，细菌组装针状结构，但毒素在与宿主细胞接触之前不会通过它分泌。细菌如何在细胞接触时激活分泌系统？同样重要的是，如果它们关闭了，它们将如何关闭？这些问题是我们理解这一制度的基础，这些问题也是本提案的重点。我们研究耶尔森氏菌III型分泌系统是因为它是一个成熟的模型系统。我们的新工具和方法揭示了III型分泌调控的核心。特别是，我们发现YopK是一种III型分泌调节因子，具有两种截然不同的调节功能，并在两个“针”的两端发挥作用。我认为YopK是分泌开关的一个组成部分。在细菌方面，YopK帮助监测哪些蛋白质是通过针头分泌的。YopK本身是分泌的，一旦进入宿主细胞，YopK就会与针状复合体的末端相互作用，关闭毒素的分泌。在这个提案中，我们将调查YopK是如何执行这些活动的。结合遗传学、细胞生物学和生物化学，我们将确定YopK如何与针两边的其他已知分泌调节因子协调。我们还将确定和描述YopK内调解其活动的功能域。总而言之，我们的工作将揭示控制这一重要致命武器的基本机制。如果我们了解开关的性质，我们就可以设计一种新型的广谱药物，针对各种细菌中的重要分泌系统。

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.