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

摘要 非分型流感嗜血杆菌(NTHI)是一种革兰氏阴性的鼻咽共生微生物， 介导人类呼吸道疾病的机会性病原体，如中耳炎(OM)、急性鼻窦炎、 囊性纤维化和慢性梗阻性疾病患者的慢性支气管炎、肺炎和恶化 肺部疾病。共生体必须适应各种微环境线索才能长期殖民 主人的名字。然而，破坏共生宿主的动态平衡可能会加剧疾病的发展。 发病机制是一个多因素的动态过程，始于NTHI向特权部位的迁移和 最终以细菌的生长而告终。增长依赖于多种复杂和协调的相互作用 在微生物(S)、遇到的各种微环境以及与宿主免疫的相互作用之间 效应器。细菌对抗先天免疫机制和获取必需营养素的策略是 对NTHI的发病至关重要。我的实验室的目标是增进我们对NTHI的了解 共生和致病行为，决定了决定这些行为的宿主微环境线索， 以及靶向发病机制，为开发治疗疾病的新疗法提供依据。我们定义了一个 抗菌肽(SAP)转运体的敏感性在NTHI抗病毒能力中的重要作用 宿主来源抗菌肽(AMPs)的致死作用。这种新的AMP识别、导入机制 细菌的胞质降解是NTHI在体内对抗宿主AMP致死所必需的。其他内容 我们实验室的数据支持SAP运输活动的多功能角色(S)，包括收购 必需的血红素铁。这些数据支持对ABC传输器的第一个描述，以导入多个 不同的底物。此外，有证据表明SAP转运蛋白复合体的差异组装 决定了这些独特的生理功能。因此，我们假设SAP转运蛋白渗透并 ATPase蛋白质协调组装独特的复合体，以驱动AMP分子的能量输入，以及 这种功能复合体不同于为进口额外底物(血红素-铁)而组装的复合体。我们 建议按点区分周质结合蛋白SAPA中的AMP和血红素结合部位 突变分析和确定SapA如何使用结合口袋识别结构不同的AMP(AIM 1)。我们将定义络合物组装的分子机制，底物运输的动力学，以及 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 不同底物获取对临床前OM模型中NTHI持久性的影响。这些研究 将为未来的研究提供必要的信息，以评估细菌对这些因素的适应情况 体内的微环境线索，并设计小分子多肽抑制剂或分子来阻断SAP- NTHI生存所必需的依赖功能。

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.