Haemophilus ducreyi Resistance to Antimicrobial Peptides

杜克雷嗜血杆菌对抗菌肽的耐药性

• 批准号: 7499712
• 负责人: MARGARET E BAUER
• 金额: $ 18.52万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-26 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7499712
• 关键词: Antibiotic Resistance; Antimicrobial Resistance; Area; Bacteria; Bacterial Infections; Binding; Binding Proteins; Biological Assay; Cell physiology; Cells; Chinchilla (genus); Class; Cytoplasm; Defensins; Development; Disease; Drug Efflux; Environment; Epithelial; Exhibits; Gene Cluster; Genes; Genital system; Genome; Goals; Gram-Negative Bacteria; HIV; Hemophilus ducreyi; Hemophilus influenza infection; Homologous Gene; Human; Immune response; In Vitro; Individual; Infection; Learning; Local Microbicides; Mediating; Microbe; Modeling; Mutation; Operon; Organism; Parents; Pectobacterium chrysanthemi; Peptides; Phagocytes; Phenotype; Plant Model; Play; Predisposition; Production; Proteolysis; Pump; Research; Resistance; Role; Sexually Transmitted Diseases; Skin; Specimen; Surface; System; Testing; Ulcer; Virulence; Work; antimicrobial peptide; bacterial resistance; bactericide; cathelicidin; design; efflux pump; extracellular; human disease; in vitro Assay; in vivo; keratinocyte; killings; macrophage; member; mutant; pathogen; periplasm; prevent; response; transmission process

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.

描述（由申请方提供）：杜克雷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，可促进人类免疫缺陷病毒（HIV）的传播和获得，从而促进HIV在地方性软下疳地区的传播。H. ducreyi主要存在于受感染个体的皮肤中，在那里它被吞噬细胞包围，但仍在细胞外。对病原体的先天性免疫应答的一个关键组成部分是杀菌性抗微生物肽（AP）的产生。在人皮肤的细菌感染期间，驻留的角质形成细胞和浸润的吞噬细胞分泌几种AP，包括α-防御素、β-防御素和凯萨林菌素LL 37到细胞外环境中。对AP杀伤的抗性是人类病原体的重要毒力机制，但在H. ducreyi。本项目的长期目标是了解H。ducreyi用来抵抗被AP杀死的东西。我们的初步研究表明，至少有一类AP，？-防御素存在于天然的软下疳样溃疡中。检测H. ducreyi对人AP的作用，我们开发了AP杀菌测定。在本试验中，H. ducreyi抵抗了在人类感染期间该生物体应该遇到的几种AP的杀死。AP抗性表型在两种已知的H. ducreyi菌株。用来自H. ducreyi感染表明H. ducreyi在感染期间表达两种转运系统，其赋予对其它细菌病原体中的AP的抗性。一种表达的转运蛋白是Sap（对抗菌肽敏感）流入泵，另一种是MTR（多重可转移抗性）流出泵。我们推测Sap和MTR转运系统参与了H. ducreyi。在目标1中，我们将在每个转运系统的一个基因中产生同基因无效突变体，并测试突变对AP易感性的影响。为了检查表达Sap和MTR两者的潜在累加效应，我们还将产生缺乏两个泵的表达的双突变体，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H. ducreyi感染目前正在研究AP在治疗中的效用，例如旨在预防包括软下疳在内的性传播感染的局部杀微生物剂。这里提出的工作是理解H. ducreyi与AP相互作用并抵抗AP的杀伤，将为开发包括AP的预防性治疗提供重要信息。 杜克雷嗜血杆菌引起软下疳，是一种重要的病原体，因为它能够促进艾滋病毒的传播，并与地方性软下疳地区艾滋病毒的传播密切相关。这项工作将有助于确定参与抗H. ducreyi对人类抗菌肽的杀伤作用，并将确定这些基因在人类感染过程中的毒力作用。抗菌肽，杀死许多病原体，正在研究其在预防性治疗针对性传播感染的效用;从这些研究中了解到的信息将是重要的开发这样的疗法。