Haemophilus ducreyi Resistance to Antimicrobial Peptides

描述(申请人提供)：杜雷氏嗜血杆菌是下巴的病原体，这是一种性传播的生殖器溃疡疾病，有助于人类免疫缺陷病毒(HIV)的传播和获得，从而导致艾滋病毒在地方性下巴流行地区的传播。杜氏杆菌主要存在于感染者的皮肤中，周围有吞噬细胞，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(AP)。在细菌感染人体皮肤的过程中，驻留的角质形成细胞和浸润性吞噬细胞会分泌多种AP进入细胞外环境，包括α-防御素、β-防御素和放线菌素LL37。抗APs致死是人类病原菌致病的重要机制之一，但在杜氏嗜血杆菌中尚未见报道。这个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，存在于自然的下巴溃疡中。为了检测杜氏嗜血杆菌对人AP的敏感性，我们建立了AP杀菌试验。在这项试验中，杜氏嗜血杆菌抵抗了几个AP的杀灭，这是该有机体在人类感染期间应该遇到的。已知的两类杜氏嗜血杆菌对AP的抗性表型相似。用人类感染杜氏嗜血杆菌模型的标本进行的体内表达研究表明，杜氏嗜血杆菌在感染过程中表达两个运输系统，使其对其他细菌病原体中的AP产生抗性。一个表达的转运体是SAP(对抗菌肽敏感)内流泵，另一个是MTR(多重可转移阻力)外排泵。我们推测SAP和MTR运输系统参与了杜氏嗜血杆菌对AP的抗性和毒力。在提案的目标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，并测试这些突变对AP易感性的影响。为了检验同时表达SAP和MTR的潜在相加效应，我们还将产生一个同时缺乏这两个泵的双重突变体，并测试其对AP介导的杀伤的敏感性。在目标2中，我们将通过在杜氏杆菌感染的人类模型中测试目标1中产生的突变株，以及母株，来测试SAP和MTR在人类感染期间的毒力中的作用。目前正在研究AP在治疗中的效用，例如旨在防止性传播感染(包括下巴)的局部杀微生物剂。这里提出的工作代表了理解H.ducreyi如何与AP相互作用并抵抗AP的杀戮的第一步，并将为包括AP在内的预防性疗法的开发提供重要信息。 杜热氏嗜血杆菌是一种重要的病原体，它能促进艾滋病毒的传播，而且它与艾滋病病毒在下巴流行地区的传播密切相关，所以它是一种重要的病原体。本申请中的工作将定义与杜氏嗜血杆菌对人类抗菌肽杀伤的抗性有关的基因，并将确定这些基因在人类感染期间毒力中的作用。抗菌肽可以杀死许多病原体，目前正在研究其在对待性传播感染的预防疗法中的效用；从这些研究中获得的信息将对开发此类疗法非常重要。

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.