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.

概括 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 它允许与真核细胞通讯以破坏正常的细胞功能。 T3SS 必不可少 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）、 志贺氏菌属（痢疾），沙门氏菌属。 （腹泻/肠热）、百日咳博德特氏菌（百日咳）和 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 与它们传递的效应蛋白相比，T3SS 装置 (T3SA) 的整体结构非常保守 和建筑。 T3SA 由以下部分组成：1) 识别分泌的细胞质分选平台 底物和分泌能力； 2）跨越整个细胞膜的刚性基体； 3）暴露的 带有尖端复合体的针，可将货物输送到宿主细胞中。分选平台的蛋白质成分有 已被识别，但在我们发表第一个描述之前，人们对它们的组装方式知之甚少。 原位志贺氏菌T3SA分选平台。从那时起，我们确定了这个结构的细节和动力学 平台，包括必需的蛋白质-蛋白质接触。基于这些背景信息，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，我们可以推断我们的发现来识别同源相互作用的抑制剂 在关系较远的 T3SA 分选平台内，例如形成铜绿假单胞菌的平台。在这里，我们将目标 内膜嵌入的 MxiG（通过其胞质结构域 MxiGc）与其新鉴定的之间的相互作用 志贺氏菌中的结合伴侣 MxiK。来自铜绿假单胞菌的同源对（分别为 PscDc 和 PscK）将 也有针对性。该试点项目应用的具体目标是：1）完成高通量筛选 扩展化学库来识别干扰 MxiGc (PscDc) 与 MxiK (PscK)； 2) 通过执行验证 MxiG-MxiK 和 PscD-PscK 相互作用抑制剂的命中 配体结合实验并确定其对志贺氏菌和假单胞菌 T3SS 相关毒力的影响 功能。该探索性项目的高风险、高回报潜力将采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测定方面 堪萨斯大学将产生协同作用，使人们对科学的理解达到前所未有的水平 T3SA 分拣平台运行的机制。