Mechanisms of Sap-mediated Antimicrobial Peptide Resistance

描述（由申请人提供）：抗菌肽（APs），包括防御素和抗菌肽，是先天免疫系统对抗细菌和其他微生物入侵者的重要组成部分。许多革兰氏阴性病原体利用对抗菌肽（Sap）敏感的转运体在感染期间抵御APs的攻击。Sap转运体已被证明对多种APs具有抗性，并对毒力有重要贡献。因此，Sap转运体是靶向开发新型抗菌药物的极好候选物，可能对许多革兰氏阴性病原体有效。然而，对于转运蛋白如何与ap一起发挥作用以抵抗其杀伤作用，人们知之甚少。该项目的长期目标是了解Sap转运体的作用机制，以便开发针对该转运体的新型抗菌药物。利用杜氏嗜血杆菌的汁液转运体作为模型系统，我们已经开始研究特定的汁液蛋白在赋予AP抗性中的作用。ducreyi是软下疳的病原体，软下疳是一种性传播的生殖器溃疡疾病，促进人类免疫缺陷病毒（HIV）的传播和获得，并有助于艾滋病毒在地方性软下疳地区的传播。杜氏嗜血杆菌是为数不多的适合人接种实验的病原体之一，因此对Sap转运体及其与APs相互作用的研究可以直接扩展到人类疾病。我们关于H. ducreyi的Sap转运体的初步数据提出了关于Sap转运体活性机制的拟议模型的问题，包括质周肽结合成分SapA在转运体活性中的作用，以及对某些APs转运体特异性的定义。在本提案中，我们将建立方法来直接检查Sap转运蛋白对人抗菌肽AP LL37的影响，并使用这些方法来确定SapA在转运蛋白活性中的作用，定义可能参与转运蛋白功能的其他蛋白质，并确定转运蛋白的哪些成分仅赋予某些APs的转运特异性。这些研究的结果为研究Sap转运体的作用机制提供了必要的工具，并将定义在Sap介导的转运中与APs特异性相互作用的蛋白质库。因此，该提案的数据将为R01应用提供方法和初步数据，以更详细地研究Sap转运体与APs的相互作用，这对于开发针对这一重要毒力因子的抗菌剂是必要的。

DESCRIPTION (provided by applicant): Antimicrobial peptides (APs), including defensins and cathelicidins, are an important component of the innate immune system for combating bacteria and other microbial invaders. Many Gram-negative pathogens utilize the Sensitive to Antimicrobial Peptides (Sap) transporter to survive attack by APs during infection. The Sap transporter has been shown to confer resistance to a wide variety of APs and contributes significantly to virulence. Thus, the Sap transporter is an excellent candidate for targeting development of novel antimicrobial therapeutics that would likely be effective against many Gram-negative pathogens. However, little is known about how the transporter functions with APs to confer resistance to their killing effects. The long-term goal of this project is to understand the mechanism of action of the Sap transporter so that novel antimicrobial agents can be developed that target this transporter. Using the Sap transporter of Haemophilus ducreyi as a model system, we have begun to study the role of specific Sap proteins in conferring AP resistance. H. ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcer disease that facilitates transmission and acquisition of human immunodeficiency virus (HIV) and contributes to the spread of HIV in areas with endemic chancroid. H. ducreyi is one of few pathogens amenable to human inoculation experiments, so studies of the Sap transporter and its interactions with APs can be directly extended to human disease. Our preliminary data with the Sap transporter of H. ducreyi raise questions about the proposed model of the mechanism of Sap transporter activity, including the role of the periplasmic peptide binding component, SapA, in transporter activity and in defining the specificity of the transporter for certain APs. In this proposal, we will establish methods to directly examine the effects of the Sap transporter on the human cathelicidin AP LL37 and use these methods to determine the role of SapA in transporter activity, define possible additional proteins involved in transporter function, and determine which components of the transporter confer specificity for transport of only certain APs. Outcomes of these studies provide the tools required to study the mechanism of action of the Sap transporter and will define the repertoire of proteins that interact specifically with APs for Sap-mediated transport. Data from this proposal will thus provide the methodologies and preliminary data for an R01 application to study in greater detail the interactions of the Sap transporter with APs, which is necessary for developing antimicrobials that target this important virulence factor.