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: 10092919
• 负责人: Kevin M Mason
• 金额: $ 50.01万
• 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 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10092919
• 关键词: ATP phosphohydrolase; ATP-Binding Cassette Transporters; Acute; Affinity; Airway Disease; Animal Model; Behavior; Binding; Binding Sites; Biochemical; Chronic Bronchitis; Chronic Obstructive Airway Disease; Complex; Coupled; 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; 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; Structure; Testing; Therapeutic; antimicrobial peptide; bactericide; combat; commensal microbes; cystic fibrosis patients; design; experience; host colonization; in vivo; infectious disease model; inhibitor/antagonist; innate immune mechanisms; migration; mutant; novel; novel therapeutics; nutrition; opportunistic pathogen; pathogenic microbe; permease; protein complex; 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.

抽象的 不可用的流感嗜血杆菌（NTHI）是一种革兰氏阴性鼻咽相关微生物，并且 介导人类气道疾病的机会性病原体，例如中耳炎（OM），急性鼻窦炎， 囊性纤维化和慢性阻塞性患者的慢性支气管炎，肺炎和加重 肺部疾病。共生必须适应各种微环境线索以进行长期定植 主持人。然而，共生主持稳态的破坏可以增强疾病的发展。 发病机理是一个多因素和动态过程，始于NTHI迁移到特权站点， 最终以细菌生长为顶。增长取决于多个复杂和协调的相互作用 在微生物之间，遇到各种微环境以及与宿主免疫的相互作用 效应子。挫败先天免疫机制和获得必需营养素的细菌策略是 对nTHI发病机理至关重要。我实验室的目标是提高我们对NTHI的理解 共生和致病行为，确定决定这些行为的宿主微环境线索， 以及发育新疗法以治疗疾病的发病机理的目标机制。我们定义了一个 敏感性对抗菌肽（SAP）转运蛋白的敏感性在NTHI的能力中的重要作用 宿主衍生的抗菌肽（AMP）的致命作用。这种新颖的AMP识别机制，导入 细菌细胞质降解对于NTHI对抗体内宿主AMP致死性至关重要。额外的 我们实验室的数据支持SAP运输活动的多功能角色，包括获取 必不可少的血红素铁。这些数据支持ABC运输蛋白的第一个描述以导入多个 不同的底物。此外，证据表明SAP转运蛋白复合蛋白的差分组装 决定了这些独特的生理功能。因此，我们假设SAP转运蛋白渗透酶和 ATPase蛋白坐标的独特复合物的组装以驱动AMP分子的能量进口，并且 该功能复合物与组装的不同之处在于进口其他底物（血红素铁）。我们 提议在周围结合蛋白SAPA中区分AMP和血红素结合位点 突变分析并确定SAPA如何使用结合口袋来识别结构上不同的放大器（AIM 1）。我们将定义复杂组装的分子机制，底物转运的动力学和 研究这些过程在细菌营养中的作用（AIM 2）。作为两个目标的一部分，我们将验证 OM临床前模型中差分底物采集对NTHI持久性的影响。这些研究 将为将来的研究提供必要的信息，以评估细菌适应性的响应 体内微环境提示，并设计小分子肽抑制剂或分子以阻断SAP- 依赖性功能对于NTHI生存至关重要。