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抵抗力的全球了解。对于 NTHI 来说，了解这一过程可以解释这种生物体如何作为共生微生物持续存在于宿主呼吸道中，同时又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 发病机制将增强中耳炎 (OM) 等疾病的健康相关研究。我们最近表明，Sap（抗菌肽敏感性）转运系统是 NTHI 的主要毒力决定因素，是抵抗抗菌肽 (AP) 杀伤作用所必需的，并且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免受宿主 AP 的透化。本应用的目的是：1) 确定 AP 是否以 Sap 依赖性方式转运至细菌细胞质，并确定转运的 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 复合物结合以介导钾吸收。我们建议测量三种不同类别的 AP 向整个细胞的转运，从而定义 Sap 转运蛋白在 AP 摄取中的功能。为了确认全细胞转运，我们将通过免疫金电子显微镜定位细胞中的 AP。然后，我们将通过反相 HPLC 分析监测全细胞裂解物细胞质部分中的肽含量和 AP 浓度，从而确定转运的 AP 的命运。在目标 2 中，我们将确定 NTHI 中的高速率钾转运是否需要 TrkH，并通过化学交联和亲和纯化监测 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可以改变 AP 通过 Sap 转运蛋白的转运。这些研究产生的数据将首次具体定义 NTHI 中 SAP 运输系统的分子机制，也许更重要的是，提供有关 NTHI 如何介导体内宿主先天免疫成分的生存的见解。 汁液转运蛋白在中耳致病细菌中提供关键且必要的功能，并介导宿主免疫反应攻击时的生存。从实现这些目标中获得的信息将增进我们对这种细菌如何引起疾病的理解，并更好地帮助我们设计对抗中耳感染的治疗方法。

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.