NMR studies of bacterial needle and tip proteins

描述（由申请方提供）：拟定研究的目的是了解细菌III型分泌物针头装置组装中涉及的蛋白质-蛋白质相互作用。这种装置类似于细菌表面的注射器，被许多病原体用于将毒力因子注入靶细胞以引发人类疾病。针状组装体由大约120个相同蛋白质的拷贝组成，这些蛋白质以超螺旋的方式排列，并且该针的顶端被尖端蛋白质覆盖。在过去的两年中，我们已经完成了两个针单体的NMR结构：BsaL从类鼻疽伯克霍尔德菌，一种与生物战相关的病原体，和PrgI从鼠伤寒沙门氏菌，一种与食物中毒相关的病原体。我们还使用NMR来确定福氏志贺菌针和尖蛋白之间的结合相互作用的关键残基。我们的目标是阐明针装置是如何组装的，通过确定精确的蛋白质-蛋白质接触涉及针和尖端蛋白质。我们将结合联合收割机核磁共振与电子显微镜和分子建模，以确定组装针的结构。我们的目标是：（#1）确定针是如何组装的，（#2）确定尖端蛋白如何对接在针上，以及（#3）使用诱变和微生物测定将结构结果与针组装和体内毒力相关联。许多病原体依赖于针头设备感染全世界数百万人，许多病原体是生物恐怖主义的潜在代理人。由于该装置暴露在细菌表面上，因此破坏针组件是开发新型抗感染药物的有吸引力的方法。这种方法需要详细了解针头组装中涉及的蛋白质-蛋白质相互作用。公共卫生相关性许多抗生素耐药性细菌病原体的出现对公共卫生和安全构成了重大问题，其中许多细菌依赖于针状蛋白质组装体感染其宿主。拟议的研究旨在通过确定其组分的蛋白质-蛋白质相互作用来阐明这种针是如何组装的。这些知识将用于设计新型抗菌剂。

DESCRIPTION (provided by applicant): The objective of the proposed studies is to understand the protein-protein interactions involved in the assembly of the bacterial type III secretion needle apparatus. This apparatus resembles a syringe on the bacterial surface and is used by many pathogens to inject virulence factors into target cells to initiate human diseases. The needle-like assembly consists of about 120 copies of identical proteins that are arranged in a superhelical manner, and the apex of this needle is capped by tip proteins. Over the past two years, we have completed the NMR structures of two needle monomers: BsaL from Burkholderia pseudomallei, a pathogen associated with biowarfare, and PrgI from Salmonella typhimurium, a pathogen associated with food poisoning. We have also used NMR to identify the key residues involved in the binding interaction between the needle and tip proteins of Shigella flexneri. Our goal is to elucidate how the needle apparatus is assembled by determining the precise protein-protein contacts involving the needle and tip proteins. We will combine NMR with electron microscopy and molecular modeling to determine the structure of the assembled needle. Our aims are: (#1) determine how the needle is assembled, (#2) determine how the tip protein docks on the needle, and (#3) use mutagenesis and microbiological assays to correlate the structural results to needle assembly and virulence in vivo. Many pathogens rely on the needle apparatus to infect millions of people worldwide and many are potential agents of bioterrorism. Because this apparatus is exposed on the bacterial surface, disrupting the needle assembly is an attractive approach for the development of novel anti-infectives. This approach requires a detailed understanding of the protein-protein interactions involved in needle assembly. PUBLIC HEALTH RELEVANCE The appearance of many antibiotic resistant bacterial pathogens poses a major problem in public health and safety and many of these bacteria rely on a needle-like protein assembly to infect their hosts. The proposed research seeks to elucidate how this needle is assembled by determining the protein-protein interactions of its components. This knowledge will then be used in the design of novel anti-bacterial agents.