The NTHI Sap Transporter: A Mechanism of Antimicrobial Peptide Resistance

描述(由申请人提供)：这项申请的广泛、长期目标是促进我们对非分型流感嗜血杆菌(NTHI)如何介导对宿主免疫反应成分的抵抗的全球理解。对NTHI来说，了解这一过程可能解释这种细菌如何作为共生微生物留在宿主呼吸道，同时保持导致上呼吸道和下呼吸道疾病的能力。显然，更好地了解NTHI的发病机制将加强对中耳炎(OM)等疾病的健康相关研究。我们最近发现SAP(对抗菌肽的敏感性)转运蛋白系统是NTHI的一个主要毒力决定因素，是抵抗抗菌肽(APs)杀死所必需的，并在该微生物的钾吸收中发挥作用。我们假设SAP蛋白保护细菌细胞质膜不被宿主AP通透。这项应用的目的是：1)确定AP是否以SAP依赖的方式运输到细菌细胞质，并确定运输的AP是否随后被细菌肽酶降解；2)确定NTHI SAPD ATPase是否直接与TrkA/H复合体结合以介导钾的吸收。我们建议测量三种不同类别的AP进入整个细胞的转运，从而确定SAP转运体在AP摄取中的功能。为了确定整个细胞的转运，我们将用免疫金电镜法定位AP在细胞中的位置。然后，我们将通过反相高效液相分析，通过监测全细胞裂解物细胞质部分的多肽含量和AP浓度来确定转运的AP的命运。在目标2中，我们将确定TrkH是否是NTHI中高速钾运输所必需的，并通过化学交联和亲和纯化来监测SAPD与TrkA/H蛋白复合体的相互作用。最后，我们将确定快速的钾摄取是否可以改变AP通过SAP转运体的运输。这些研究产生的数据将首次明确NTHI中SAP运输系统的分子机制，或许更重要的是，有助于深入了解NTHI如何在体内调节宿主固有免疫成分的生存。 SAP转运蛋白在中耳致病细菌中起着关键和必要的作用，并在宿主免疫反应的攻击下调节生存。从实现这些目标中获得的信息将促进我们对这种细菌如何导致疾病的理解，并更好地为我们设计抗击中耳感染的治疗方法提供装备。

DESCRIPTION (provided by applicant): The broad, long-term objective of this application is to further our global understanding of how nontypeable Haemophilus influenzae (NTHI) mediates resistance to components of the host immune response. For NTHI, understanding this process may explain how this organism persists in the host airway as a commensal microorganism and yet maintains the ability to cause disease of both the upper and lower respiratory tracts. Clearly, a better understanding of NTHI pathogenesis will augment health related research in diseases such as otitis media (OM). We have recently shown that the Sap (sensitivity to antimicrobial peptides) transporter system is a major virulence determinant of NTHI, is required for resistance to killing by antimicrobial peptides (APs) and plays a role in potassium uptake in this microorganism. We hypothesize that the Sap proteins protect the bacterial cytoplasmic membrane from permeabilization by host APs. The aims of this application are to: 1) Determine whether APs are transported to the bacterial cytoplasm in a Sap-dependent manner, and determine whether transported APs are then degraded by bacterial peptidases and 2) Determine whether the NTHI SapD ATPase directly associates with the TrkA/H complex to mediate potassium uptake. We propose to measure transport of three distinct classes of APs into whole cells and thereby define the function of the Sap transporter in AP uptake. In order to confirm whole-cell transport we will localize APs in cells by immunogold electron microscopy. We will then determine the fate of transported APs by monitoring peptide contents and AP concentrations in the cytoplasmic fraction of whole cell lysates by reverse-phase HPLC analysis. In Aim 2, we will determine whether TrkH is required for high rate potassium transport in NTHI and monitor the interaction of SapD with the TrkA/H protein complex by chemical cross-linking and affinity purification. Finally, we will determine whether rapid potassium uptake can alter AP transport via the Sap transporter. The data generated from these studies will specifically define, for the first time, the molecular mechanism of the Sap transport system in NTHI and, perhaps more importantly, provide insight into how NTHI mediates survival in response to host innate immune components in vivo. The Sap transporter provides a critical and necessary function in disease-causing bacteria of the middle ear, and mediates survival in response to attack by the host immune response. The information gained from fulfillment of these aims will advance our understanding of how this bacterium causes disease and better equip us to design therapeutic approaches to combat middle ear infections.