Identification of small molecule probes for dissecting the roles of sorting platform components within the type III secretion system

摘要 我们的实验室研究细菌III型分泌系统(T3SS)，这是一种复杂的纳米机器 这允许与真核细胞进行通信，以颠覆正常的细胞功能。T3SS是必不可少的 一些生物医学上重要的人类病原体的毒力因子，包括鼠疫耶尔森氏菌(鼠疫)， 志贺氏菌属(痢疾)，沙门氏菌属。(腹泻/肠热病)，百日咳波氏杆菌(百日咳)，以及 铜绿假单胞菌(机会性/医院感染)。虽然这些系统在以下方面是不同的 它们传递的效应蛋白，T3SS装置(T3SA)在整体结构上非常保守 和建筑。T3SA包括：1)识别分泌物的细胞质分选平台 底物和分泌物；2)覆盖整个细胞膜的坚硬的基底；以及3)暴露的 带有尖端复合体的针头，将货物输送到宿主细胞。分拣平台的蛋白质组分有 已经被确认，但在我们发表了对In的第一个描述之前，人们对它们是如何组装的知之甚少 原位志贺氏菌T3SA分选平台。从那时起，我们已经确定了这一结构的细节和动态 平台，包括必需的蛋白质-蛋白质接触。基于这一背景信息，我们 假设分选平台内的关键接口可以靶向阻断III型分泌 志贺氏菌。此外，我们可以根据我们的发现来确定同源相互作用的抑制因素。 在远亲的T3SA分选平台内，例如形成铜绿假单胞菌。在这里，我们将针对 内膜包埋MxiG(通过其细胞质结构域，MxiGc)与其新鉴定的 志贺氏菌中的结合伙伴MxiK。来自铜绿假单胞菌的同源对(分别为PscDc和PscK)将 也要成为目标。该试点项目应用的具体目标是：1)完成高通量筛分 用于识别干扰MxiGc(PscDc)与MxiGc相互作用的小分子 MxiK(PscK)；以及2)通过执行以下操作验证MxiG-MxiK和PscD-PscK相互作用的抑制剂的HITS 配体结合实验及其对志贺氏菌和假单胞菌T3SS毒力的影响 功能。这个探索性项目的高风险、高回报潜力将使用专门的核心实验室 在高通量筛选、计算化学生物学和蛋白质结构确定方面 堪萨斯大学将产生协同效应，使人们能够前所未有地了解 T3SA分拣平台的运行机制。

SUMMARY Our laboratory studies the bacterial type III secretion system (T3SS) which is a sophisticated nanomachine that permits communication with eukaryotic cells to subvert normal cellular functions. T3SSs are essential virulence factors for a number of biomedically important human pathogens including Yersinia pestis (plague), Shigella spp. (dysentery), Salmonella spp. (diarrhea/enteric fever), Bordetella pertussis (whooping cough), and Pseudomonas aeruginosa (opportunistic/nosocomial infections). While these systems are diverse with regard to the effector proteins they deliver, the T3SS apparatus (T3SA) is well-conserved with respect to overall structure and architecture. The T3SA is comprised of: 1) a cytoplasmic sorting platform that recognizes secretion substrates and powers secretion; 2) a rigid basal body spanning the entire cell envelope; and 3) an exposed needle with tip complex that delivers cargo into host cells. The protein components of the sorting platform have been identified, but little was known about how they assemble until we published the first description of the in situ Shigella T3SA sorting platform. Since then, we have determined the structural details and dynamics of this platform, including the essential protein-protein contacts. Based on this background information, we hypothesize that key interfaces within the sorting platform can be targeted to block type III secretion in Shigella. Furthermore, we can extrapolate our findings to identify inhibitors of homologous interactions within distantly related T3SA sorting platforms such as that form P. aeruginosa. Here, we will target the interaction between inner membrane-imbedded MxiG (via its cytoplasmic domain, MxiGc) and its newly identified binding partner, MxiK, in Shigella. The homologous pair from P. aeruginosa (PscDc and PscK, respectively) will also be targeted. The specific aims of this pilot project application are to: 1) Complete high-throughput screens of extended chemical libraries to identify small molecules that interfere with the interaction of MxiGc (PscDc) with MxiK (PscK); and 2) Validate hits for inhibitors of the MxiG-MxiK and PscD-PscK interactions by performing ligand binding experiments and determining their effects on Shigella and Pseudomonas T3SS-related virulence functions. This high-risk, high-payoff potential of this exploratory project will employ specialized core laboratories in high-throughput screening, computational chemical biology and protein structure determination at the University of Kansas to generate a synergy that will allow an unprecedented level of understanding of the mechanism by which the T3SA sorting platform operates.