Haemophilus ducreyi Resistance to Antimicrobial Peptides

描述（由申请人提供）：杜氏嗜血杆菌是软下疳的病原体，软下疳是一种性传播的生殖器溃疡疾病，容易传播和获得人类免疫缺陷病毒（HIV），从而导致艾滋病毒在地方性软下疳地区传播。杜氏嗜血杆菌主要存在于感染者的皮肤中，被吞噬细胞包围，但仍留在细胞外。对病原体的先天免疫反应的一个关键组成部分是产生杀菌抗菌肽（APs）。在人体皮肤细菌感染过程中，常驻角质形成细胞和浸润性吞噬细胞分泌多种APs，包括a-防御素、¿-防御素和抗菌肽LL37等，进入细胞外环境。对APs杀伤的抗性是人类致病菌的一个重要毒力机制，在杜氏嗜血杆菌中尚未得到研究。该项目的长期目标是了解H. ducreyi用来抵抗ap杀死的机制。我们的初步研究表明，至少有一类ap，即？-防御素，存在于天然的软膜溃疡中。为了检测杜氏嗜血杆菌对人类AP的敏感性，我们建立了一种AP杀菌试验。在这个实验中，杜氏芽胞杆菌抵抗了人类感染过程中应该遇到的几种APs的杀伤。在已知的两类杜氏嗜血杆菌菌株中，AP抗性表型相似。人杜克雷伊氏杆菌感染模型的体内表达研究表明，杜克雷伊氏杆菌在感染过程中表达两种转运系统，使其对其他细菌病原体中的APs具有耐药性。一种表达的转运蛋白是Sap（对抗菌肽敏感）内流泵，另一种是MTR（多重可转移耐药）外流泵。我们假设Sap和MTR运输系统有助于杜氏弧菌对AP的抗性和毒力。在该提案的目标1中，我们将在每个运输系统的一个基因中产生等基因零突变，并测试突变对APs易感性的影响。为了检验表达Sap和MTR的潜在加性效应，我们还将产生一个缺乏这两种泵的双突变体，并测试其对ap介导的杀伤的易感性。在第二期研究中，我们将通过在杜氏嗜血杆菌感染的人类模型中测试第二期研究中产生的突变体以及亲本菌株，来测试Sap和MTR在人类感染期间的毒力作用。目前，人们正在研究APs在治疗中的应用，如局部杀微生物剂，用于预防包括软下疳在内的性传播感染。这里提出的工作代表了了解H. ducreyi如何与ap相互作用并抵抗ap杀伤的第一步，并将为开发包括ap在内的预防性治疗提供重要信息。

DESCRIPTION (provided by applicant): Haemophilus ducreyi is the causative agent of chancroid, a sexually transmitted genital ulcer disease that facilitates transmission and acquisition of human immunodeficiency virus (HIV) and thus contributes to the spread of HIV in areas with endemic chancroid. H. ducreyi resides primarily in the skin of infected individuals, where it is surrounded by phagocytes but remains extracellular. A key component of the innate immune response to pathogens is production of bactericidal antimicrobial peptides (APs). During bacterial infection of human skin, resident keratinocytes and infiltrating phagocytes secrete several kinds of APs, including a- defensins, ¿-defensins, and cathelicidin LL37, into the extracellular milieu. Resistance to killing by APs is an important virulence mechanism of human pathogens that has not been studied in H. ducreyi. The long-term goal of this project is to understand mechanisms H. ducreyi uses to resist being killed by APs. Our preliminary studies showed that at least one class of APs, the ?-defensins, are present in natural chancroidal ulcers. To examine susceptibility of H. ducreyi to human APs, we developed an AP bactericidal assay. In this assay, H. ducreyi resisted killing by several APs that the organism should encounter during human infection. The AP resistance phenotype was similar in the two known classes of H. ducreyi strains. In vivo expression studies with specimens from the human model of H. ducreyi infection demonstrated that H. ducreyi expresses two transport systems during infection that confer resistance to APs in other bacterial pathogens. One expressed transporter is a Sap (sensitive to antimicrobial peptides) influx pump, and the other is an MTR (multiple transferable resistance) efflux pump. We hypothesize that the Sap and MTR transport systems contribute to AP resistance and virulence of H. ducreyi. In Aim 1 of the proposal, we will generate isogenic null mutants in one gene of each transport system and test the effects of the mutations on susceptibility to APs. To examine potential additive effects of expressing both Sap and MTR, we will also generate a double mutant lacking expression of both pumps and test its susceptibility to AP-mediated killing. In Aim 2, we will test the role of Sap and MTR in virulence during human infection by testing the mutants generated in Aim 1, alongside the parent strain, in the human model of H. ducreyi infection. APs are currently being studied for their utility in therapies such as topical microbicides designed to prevent sexually transmitted infections including chancroid. The work proposed here represents the first step in understanding how H. ducreyi interacts with and resists killing by APs and will provide important information for development of preventative therapies that include APs. Haemophilus ducreyi, which causes chancroid, is an important pathogen because of its ability to facilitate HIV transmission and its strong correlation with the spread of HIV in areas with endemic chancroid. The work in this application will define genes involved in the resistance of H. ducreyi to killing by human antimicrobial peptides and will determine the roles of these genes in virulence during human infection. Antimicrobial peptides, which kill many pathogens, are being studied for their utility in preventative therapies targeting sexually transmitted infections; the information learned from these studies will be important for developing such therapies.