A novel multifunctional role of diverse substrate binding and import by the Haemophilus Sap transporter

嗜血杆菌汁液转运蛋白多种底物结合和输入的新型多功能作用

• 批准号: 10570899
• 负责人: Kevin M Mason
• 金额: $ 45.48万
• 依托单位: RESEARCH INST NATIONWIDE CHILDREN'S HOSP
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10570899
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Acute; Affinity; Airway Disease; Animal Model; Behavior; Binding; Binding Sites; Biochemical; Chronic Bronchitis; Chronic Obstructive Pulmonary Disease; Complex; Coupled; Crystallography; Cues; Cytoplasm; Data; Defense Mechanisms; Development; Disease; Future; Goals; Growth; Haemophilus influenza virulence; Heme; Heme Iron; Hemophilus; Hemophilus influenza infection; Homeostasis; Human; Immune; Infection; Investigation; Kinetics; Knowledge; Laboratories; Lower respiratory tract structure; Mediating; Metabolic; Microbe; Molecular; Mutation; Nasopharynx; Nontypable Haemophilus influenza; Nutrient; Nutritional; Otitis Media; Outcome; Pathogenesis; Pathogenicity; Peptide Transport; Peptides; Periplasmic Binding Proteins; Physiological; Pneumonia; Positioning Attribute; Pre-Clinical Model; Process; Proteins; RNA Interference; Resistance; Role; Sinusitis; Site; Specificity; Testing; Therapeutic; Upper respiratory tract; antimicrobial peptide; bactericide; combat; commensal microbes; cystic fibrosis patients; design; experience; host colonization; in vivo; infectious disease model; inhibitor; innate immune mechanisms; migration; mutant; novel; novel therapeutics; nutrition; opportunistic pathogen; pathogenic microbe; permease; protein complex; rational design; response; small molecule; small molecule inhibitor; uptake

Abstract Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative nasopharyngeal commensal microbe, and opportunistic pathogen that mediates human airway diseases such as otitis media (OM), acute sinusitis, chronic bronchitis, pneumonia, and exacerbations in patients with cystic fibrosis and chronic obstructive pulmonary diseases. Commensals must adapt to various microenvironmental cues for long-term colonization of the host. Disruption of commensal-host homeostasis however, can potentiate disease development. Pathogenesis is a multifactorial and dynamic process that begins with NTHI migration to a privileged site and culminates with bacterial growth. Growth is dependent upon multiple complex and coordinated interactions between the microbe(s), the varied microenvironments encountered, and interactions with host immune effectors. Bacterial strategies to thwart innate immune mechanisms and acquisition of essential nutrients are critical for NTHI pathogenesis. The goals of my laboratory are to advance our understanding of NTHI commensal and pathogenic behaviors, determine host microenvironmental cues that dictate these behaviors, and target mechanisms of pathogenesis for development of novel therapies to treat disease. We defined an essential role for the sensitivity to antimicrobial peptide (Sap) transporter in the ability of NTHI to counter the lethal effects of host-derived antimicrobial peptides (AMPs). This novel mechanism of AMP recognition, import and bacterial cytoplasmic degradation is essential for NTHI to counter host AMP lethality in vivo. Additional data from our laboratory support a multi-functional role(s) for Sap transport activity, including the acquisition of essential heme-iron. These data support the first description of an ABC transporter to import more than one diverse substrate. Further, evidence indicates that differential assembly of Sap transporter complex proteins dictates these unique physiological functions. We therefore hypothesize that Sap transporter permease and ATPase proteins coordinate assembly of unique complexes to drive energetic import of AMP molecules, and that this functional complex differs from that assembled for import of additional substrates (heme-iron). We propose to differentiate the AMP and heme binding sites in the periplasmic binding protein, SapA, by point mutant analysis and determine how SapA uses the binding pocket to recognize structurally diverse AMPs (Aim 1). We will define the molecular mechanisms of complex assembly, kinetics of substrate transport, and investigate a role for these processes in bacterial nutrition (Aim 2). As part of both aims we will validate the impact of differential substrate acquisition on NTHI persistence in the preclinical model of OM. These studies will provide the necessary information for future studies to assess bacterial adaptation in response to these microenvironmental cues in vivo and to design small molecule peptide inhibitors or molecules to block Sap- dependent functions essential for NTHI survival.

摘要 无法分型的流感嗜血杆菌（NTHI）是一种革兰氏阴性鼻咽部微生物， 介导人类气道疾病如中耳炎（OM），急性鼻窦炎， 慢性支气管炎、肺炎以及囊性纤维化和慢性阻塞性肺疾病患者的病情加重 肺部疾病。共栖生物必须适应各种微环境线索才能长期定居 的主机。然而，宿主-宿主体内平衡的破坏可增强疾病的发展。 发病机制是一个多因素和动态的过程，开始于NTHI迁移到一个特权网站， 随着细菌的生长而达到高潮。增长依赖于多种复杂和协调的相互作用 微生物之间，遇到的各种微环境，以及与宿主免疫的相互作用 效应器细菌阻碍先天免疫机制和获得必需营养素的策略是 对NTHI发病机制至关重要。我的实验室的目标是促进我们对NTHI的理解 寄生和致病行为，决定宿主微环境线索，支配这些行为， 和发病机理的靶向机制，用于开发治疗疾病的新疗法。我们定义了 抗微生物肽（Sap）转运蛋白敏感性在NTHI对抗抗肿瘤能力中的重要作用。 宿主来源的抗菌肽（AMP）的致死作用。这种AMP识别的新机制， 细菌胞质降解是NTHI对抗宿主AMP体内致死性所必需的。额外 来自我们实验室的数据支持SAP运输活动的多功能作用，包括获取 必需血红素铁这些数据支持第一个描述的ABC运输进口超过一个 多样的基质此外，有证据表明，Sap转运蛋白复合物蛋白的差异组装 决定了这些独特的生理功能。因此，我们假设SAP转运蛋白通透酶和 ATP酶蛋白协调独特复合物的组装以驱动AMP分子的能量输入， 这种功能复合物不同于为输入额外底物（血红素-铁）而组装的复合物。我们 建议区分AMP和血红素结合位点的周质结合蛋白，SapA，由点 突变体分析和确定SapA如何使用结合口袋来识别结构多样的AMP（Aim 1）。我们将定义复杂组装的分子机制，底物运输的动力学， 研究这些过程在细菌营养中的作用（目标2）。作为这两个目标的一部分，我们将验证 差异底物采集对OM临床前模型中NTHI持续性的影响。这些研究 将为未来的研究提供必要的信息，以评估细菌适应这些 在体内的微环境线索，并设计小分子肽抑制剂或分子，以阻止Sap- 对NTHI存活至关重要的依赖性功能。