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

鉴定小分子探针，用于剖析 III 型分泌系统中分选平台组件的作用

• 批准号: 9806976
• 负责人: WILLIAM D. PICKING
• 金额: $ 22.73万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9806976
• 关键词: ATP phosphohydrolase; Anti-Infective Agents; Architecture; Bacteria; Binding; Binding Sites; Biology; Bordetella pertussis; Cell physiology; Cells; Chemicals; Clinical; Communication; Complex; Crystallization; Cytoplasmic Tail; Diarrhea; Distant; Dysentery; Ensure; Eukaryotic Cell; Foundations; Gram-Negative Bacteria; Heart; Homologous Protein; Human; Impairment; In Situ; In Vitro; Investigation; Kansas; Laboratories; Laboratory Study; Libraries; Ligand Binding; Mechanics; Membrane; Needles; Nosocomial Infections; Pertussis; Pilot Projects; Plague; Positioning Attribute; Proteins; Pseudomonas; Pseudomonas aeruginosa; Publishing; Recombinants; Reporting; Resolution; Role; Salmonella; Shigella; Site; Sorting - Cell Movement; Structural Protein; Structure; Structure-Activity Relationship; System; Testing; Therapeutic Agents; Time; Type III Secretion System Pathway; Typhoid Fever; Universities; Virulence; Virulence Factors; Work; Yersinia pestis; antimicrobial; base; cell envelope; experimental study; high risk; high throughput screening; human pathogen; inhibitor/antagonist; kinetosome; nanomachine; novel; pathogenic bacteria; protein protein interaction; protein structure; response; scaffold; screening; small molecule; small molecule inhibitor; small molecule libraries; stoichiometry; synergism; tool

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.

总结 我们的实验室研究细菌III型分泌系统（T3 SS），这是一种复杂的纳米机器 它能与真核细胞进行通讯从而破坏正常的细胞功能。T3 SS是必不可少的 许多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）， 志贺菌（痢疾）、沙门氏菌属（Salmonella spp.）（腹泻/肠热病）、百日咳杆菌（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各不相同： 由于它们所传递的效应蛋白，T3 SS装置（T3 SA）在整体结构上是非常保守的 和建筑。T3 SA包括：1）识别分泌物的细胞质分选平台 基质和权力的分泌; 2）刚性基体跨越整个细胞包膜;和3）暴露 具有尖端复合物的针，其将货物递送到宿主细胞中。分选平台的蛋白质组分具有 已经被确定，但很少有人知道他们如何组装，直到我们发表了第一个描述， 志贺氏菌T3 SA原位分选平台。从那时起，我们已经确定了结构细节和动力学， 平台，包括必要的蛋白质-蛋白质接触。根据这些背景信息，我们 假设分选平台内的关键界面可以靶向阻断III型分泌， 志贺氏菌此外，我们可以推断我们的研究结果，以确定抑制同源相互作用 在远亲的T3 SA分选平台中，例如形成铜绿假单胞菌的平台。在这里，我们将针对 嵌入内膜的MxiG（通过其胞质结构域，MxiGc）与其新鉴定的 结合伴侣MxiK。来自铜绿假单胞菌的同源对（分别为PscDc和PscK）将 也是有针对性的。该试点项目申请的具体目标是：1）完成高通量筛选 扩展的化学文库，以鉴定干扰MxiGc（PscDc）与 MxiK（PscK）;和2）通过进行MxiG-MxiK和PscD-PscK相互作用的抑制剂的筛选， 配体结合实验及其对志贺氏菌和假单胞菌T3 SS相关毒力的影响 功能协调发展的这个高风险、高回报的探索性项目将雇佣专业的核心实验室 在高通量筛选、计算化学生物学和蛋白质结构测定方面， 堪萨斯大学产生的协同作用，将允许前所未有的理解水平， T3 SA分拣平台运行的机制。