The NTHI Sap Transporter: A Mechanism of Antimicrobial Peptide Resistance

描述（由申请方提供）：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应答组分的耐药性的全球理解。对于NTHI，了解这一过程可以解释这种微生物如何作为呼吸道微生物在宿主气道中持续存在，但仍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发病机制将增加健康相关的疾病，如中耳炎（OM）的研究。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）杀死所必需的，并在这种微生物的钾吸收中发挥作用。我们推测，SAP蛋白保护细菌细胞质膜透化主机AP。本申请的目的是：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，以及2）确定NTHI SapD ATP酶是否直接与TrkA/H复合物缔合以介导钾摄取。我们建议测量三种不同类别的AP到整个细胞的运输，从而定义SAP转运蛋白在AP摄取中的功能。为了确认全细胞运输，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的细胞质组分中的肽含量和AP浓度来确定转运AP的命运。在目标2中，我们将确定是否TrkH是所需的高速率钾运输NTHI和监测的TrkA/H蛋白复合物的化学交联和亲和纯化的SapD的相互作用。最后，我们将确定是否快速钾的吸收可以改变AP运输通过SAP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，提供了对NTHI如何介导体内宿主先天免疫组分应答的生存的见解。 Sap转运蛋白在中耳的致病细菌中提供关键和必要的功能，并介导对宿主免疫应答攻击的存活。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引起疾病的理解，并更好地帮助我们设计治疗方法来对抗中耳感染。

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.