Mechanisms of Sap-mediated Antimicrobial Peptide Resistance

描述（由申请人提供）：抗菌肽（AP），包括防御素和cathelicidins，是先天免疫系统的重要组成部分，用于对抗细菌和其他微生物入侵者。许多革兰氏阴性病原体利用对抗菌肽（Sap）敏感的转运蛋白在感染期间抵抗AP的攻击。Sap转运蛋白已被证明赋予对多种AP的抗性，并显著促进毒力。因此，Sap转运蛋白是靶向开发可能有效对抗许多革兰氏阴性病原体的新型抗微生物治疗剂的极好候选物。然而，很少有人知道转运蛋白如何与AP一起发挥作用，以抵抗其杀伤作用。该项目的长期目标是了解Sap转运蛋白的作用机制，以便开发针对该转运蛋白的新型抗菌剂。利用杜克雷嗜血杆菌的Sap转运蛋白作为模型系统，我们已经开始研究特定的Sap蛋白在赋予AP抗性中的作用。H. 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HIV在地方性软下疳地区的传播。H. ducreyi是少数几种适合人类接种实验的病原体之一，因此Sap转运蛋白及其与AP相互作用的研究可以直接扩展到人类疾病。我们对H. ducreyi提出了关于Sap转运蛋白活性机制的拟议模型的问题，包括周质肽结合组分SapA在转运蛋白活性中的作用以及在定义某些AP的转运蛋白特异性方面的作用。在这个提议中，我们将建立方法来直接检查Sap转运蛋白对人cathelicidin AP LL 37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组分赋予仅某些AP转运的特异性。这些研究的结果提供了所需的工具来研究SAP转运蛋白的作用机制，并将定义与SAP介导的运输AP特异性相互作用的蛋白质的库。因此，来自该提案的数据将为R 01应用提供方法和初步数据，以更详细地研究SAP转运蛋白与AP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。 公共卫生相关性：许多革兰氏阴性细菌使用Sap转运蛋白来避免被身体产生的抗微生物肽杀死，以抵御入侵的微生物。在重要病原体中这种毒力因子的保守性使其成为开发新的抗微生物疗法的有吸引力的候选者;然而，这种转运蛋白的作用机制还不清楚。本申请中概述的工作将定义Sap转运蛋白如何帮助细菌在宿主中存活，并将定义所涉及的蛋白质;从这些研究中获得的信息对于开发阻断这种转运蛋白活性的新的抗微生物疗法将是重要的。

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. PUBLIC HEALTH RELEVANCE: Many gram-negative bacteria use the Sap transporter to avoid being killed by antimicrobial peptides the body produces as a defense against invading microbes. The conservation of this virulence factor among significant pathogens makes it an attractive candidate for development of new antimicrobial therapies; however, the mechanism of action of this transporter is not well understood. The work outlined in this application will define how the Sap transporter aids the bacteria in survival in the host and will define the proteins involved; information learned from these studies will be important for developing new antimicrobial therapeutics that block the activity of this transporter.