Assembly and Function of the Bacterial Type III Secretion System Basal Body

描述（由申请人提供）：蛋白III型分泌系统（T3SS）是一种超分子有机纳米机器，可将细菌毒力蛋白注入真核细胞，以调节其生理学，从而使病原体受益。它是许多医学相关生物体毒力的基础。对这一系统的机械理解是现代感染生物学的核心科学问题之一。同时，这是一个问题，其解决方案也将提供重要的转化益处，从而允许开发靶向注射装置的抗毒性化合物。此外，T3SS代表了纳米机器最迷人的例子之一。从智力上讲，研究T3SS的结构、功能和机制将影响对动植物传染病的理解，并增加对纳米技术和机械的理解。这项资助旨在解决一个具体的问题：跨越革兰氏阴性菌两层膜的T3SS的组装和功能的结构基础是什么？该项目分为三个具体目标：（1）优化和扩展T3SS注射器复合物表达和纯化的现有方案（2）开发表达和纯化分泌复合物的第一种重组方法，以及（3）通过X射线晶体学确定T3SS纳米机器的高分辨率结构。为了在分子水平上理解T3SS，需要高分辨率的结构信息。虽然中分辨率电子显微镜（EM）已经给出了系统的整体视图，但需要X射线晶体学研究来发展机械理解。这种T3SS组装的“蓝图”将是理解其功能以及如何在治疗背景下抑制这种功能的关键第一步。

DESCRIPTION (provided by applicant): The protein Type III Secretion System (T3SS) is a supramolecular, organic nanomachine that injects bacterial virulence proteins into eukaryotic cells to modulate their physiology for the benefit of the pathogen. It underlies the virulence of numerous medically relevant organisms. A mechanistic understanding of this system is one of the central scientific problems in modern infection biology. At the same time, it is a problem whose solution will also provide important translational benefits, allowing for the development of antivirulence compounds, which target the injection device. Furthermore, the T3SS represents one of the most fascinating examples of a nanomachine. Intellectually, studying the structure, function, and mechanism of the T3SS will impact the understanding of infectious diseases in animals and plants, as well as increase the understanding of nanotechnology and machineries. This grant proposes to solve a specific problem: what is the structural basis for the assembly and function of the T3SS spanning the two membranes of Gram negative bacteria? The project is divided into three specific aims: (1) Optimize and extend current protocols for the expression and purification of T3SS syringe complexes (2) Develop the first recombinant methods for expressing and purifying the secretion complex, and (3) Determine the high-resolution structure of the T3SS nanomachine by X-ray crystallography. To understand the T3SS at the molecular level, high-resolution structural information is required. While moderate resolution electron microscopy (EM) has given an overall view of the system, X-ray crystallographic studies will be required to develop a mechanistic understanding. Such a "blueprint" for the assembly of the T3SS will be a critical first step towards understanding its function and how to inhibit this function in a therapeutic context.