The NTHI Sap Transporter: A Mechanism of Antimicrobial Peptide Resistance

描述（由申请人提供）：本申请的广泛、长期目标是进一步加深我们对不可分型流感嗜血杆菌（NTHI）如何介导对宿主免疫反应成分的抗性的全球理解。对于NTHI，了解这一过程可以解释这种微生物如何作为共生微生物在宿主气道中持续存在，并保持引起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。我们最近表明，Sap（对抗菌肽的敏感性）转运系统是NTHI的主要毒力决定因素，是抵抗抗菌肽（APs）杀伤所必需的，并在这种微生物的钾吸收中起作用。我们假设Sap蛋白保护细菌细胞质膜免受宿主APs的渗透作用。本应用的目的是：1)确定APs是否以sap依赖的方式转运到细菌细胞质中，并确定转运的APs是否随后被细菌肽酶降解。2)确定NTHI SapD atp酶是否直接与TrkA/H复合物结合介导钾摄取。我们建议测量三种不同类型的AP向整个细胞的运输，从而定义Sap转运体在AP摄取中的功能。为了确认全细胞转运，我们将在免疫金电镜下定位APs在细胞中的位置。然后，我们将通过反相高效液相色谱分析监测全细胞裂解物细胞质部分的肽含量和AP浓度，从而确定转运AP的命运。在Aim 2中，我们将确定TrkH是否需要NTHI中的高速率钾运输，并通过化学交联和亲和纯化来监测SapD与TrkA/H蛋白复合物的相互作用。最后，我们将确定钾的快速吸收是否会改变AP通过Sap转运体的转运。从这些研究中产生的数据将首次明确定义NTHI中Sap转运系统的分子机制，也许更重要的是，为NTHI如何在体内介导宿主先天免疫成分的生存提供洞见。

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.