Molecular basis of Pseudomonas aeruginosa persistence in chronic wound infections

慢性伤口感染中铜绿假单胞菌持续存在的分子基础

• 批准号: 8524608
• 负责人: Soyeon Lippman
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8524608
• 关键词: Accounting; Address; Antibiotics; Attenuated; Bacteria; Biological Assay; Candidate Disease Gene; Cell physiology; Chronic; Clinical; Complex; Coupled; DNA Sequence Analysis; Development; Diabetes Mellitus; Diabetic Foot Ulcer; Diabetic mouse; Environment; Evolution; Family; Genes; Genome; Goals; Growth; Healed; Health; Healthcare; Immune response; Impairment; In Vitro; Infection; Insertion Mutation; Lead; Libraries; Microbe; Microbial Biofilms; Modality; Modeling; Molecular; Mus; Mutagenesis; Mutation; Obesity; Outcome; Oxidative Stress; Pathogenesis; Patients; Phagocytes; Phagocytosis; Pharmaceutical Preparations; Physiology; Process; Property; Proteins; Pseudomonas aeruginosa; Public Health; Quality of life; Reproducibility; Research; Resistance; Role; Sampling; Technology; Testing; Therapeutic; Update; Validation; Wound Healing; Wound Infection; antimicrobial; antimicrobial drug; base; care burden; cell motility; conventional therapy; diabetic; disability; drug discovery; drug efficacy; fitness; genome-wide; genome-wide analysis; healing; improved; in vivo; killings; mutant; new therapeutic target; next generation sequencing; pathogen; public health relevance; stem; therapeutic target; wound

DESCRIPTION (provided by applicant): Our long-term goal is to elucidate the cellular functions of Pseudomonas aeruginosa required for initiating infection and needed for persistence in chronic wounds. The findings will then be used to identify effective therapeutic targets against P. aeruginosa to promote wound healing. Despite increasing evidence pointing to the inefficient elimination of opportunistic pathogens as a major reason for why chronic wounds do not heal, the molecular mechanism underlying the role of microbes in the pathogenesis of chronic wound infections remains unclear. P. aeruginosa is a key causative agent for chronic wound infections, particularly in diabetic foot ulcers. Increasing evidence suggests bacterial existence in biofilms to impede eradication by host immune response and antimicrobial treatments. Moreover, escalating emergence of resistance to existing antibiotics, lack of new antibiotic development, and rising cases of diabetes and obesity fuel the need for improved therapeutic modalities. To address this important public health issue, comprehensive identification using next-generation sequencing and chronic wound infection model in diabetic mice will be used in these specific aims: (1) Identify P. aeruginosa functions required to initiate infection and persist in chronic wound infections. To examine mutants at genome-wide scale, a recently developed transposon-sequencing (Tn-seq) technology will be employed. Tn-seq utilizes massively parallel DNA sequence analysis to track millions of bacteria carrying unique transposon insertion mutations. A library pool containing mutations in nearly all nonessential genes will be created and used to infect mouse wounds. Mutants that fail to survive in the wound environment will be identified using Tn-seq. The gene products of identified mutants may lead to potential targets for new therapeutic treatment. (2) Validate impaired infection of candidate functions identified in Aim 1. Identified functions will be reexamined in vivo using a single mutant infection. In parallel, candidate genes will be inactivated in clinical wound isolate to test whether attenuated infectivity can still be observed in clinical strain background. (3) Functionally characterize candidate genes to help understand the molecular basis underlying attenuated infectivity. Assays will test whether functions known to be involved in infection are impaired in the mutants, including biofilm formation, motility, and resistance to killing by phagocytic cells. The anticipated outcome of this project is a comprehensive identification of genes required for P. aeruginosa infection and validated potential targets for therapeutic treatment of diabetic chronic wound infections.

描述（由申请人提供）：我们的长期目标是阐明铜绿假单胞菌启动感染所需的细胞功能，以及在慢性伤口中持续存在所需的细胞功能。这些发现将用于确定针对铜绿假单胞菌的有效治疗靶点，以促进伤口愈合。尽管越来越多的证据表明，机会致病菌的无效消除是慢性伤口不愈合的主要原因，但微生物在慢性伤口感染发病机制中的作用的分子机制仍不清楚。铜绿假单胞菌是慢性伤口感染，特别是糖尿病足溃疡的关键病原体。越来越多的证据表明，生物膜中的细菌存在会阻碍宿主免疫反应和抗菌治疗的根除。此外，对现有抗生素的耐药性不断增加，缺乏新的抗生素开发，以及糖尿病和肥胖症病例的增加，都促使人们需要改进治疗方法。为了解决这一重要的公共卫生问题，将使用下一代测序和糖尿病小鼠慢性伤口感染模型进行全面鉴定，以实现以下具体目标：（1）鉴定铜绿假单胞菌启动 感染，并持续慢性伤口感染。为了在全基因组范围内检查突变体，将采用最近开发的转座子测序（Tn-seq）技术。Tn-seq利用大规模并行DNA序列分析来跟踪数百万携带独特转座子插入突变的细菌。一个包含几乎所有非必需基因突变的文库库将被创建并用于感染小鼠伤口。将使用Tn-seq鉴定在伤口环境中不能存活的突变体。鉴定的突变体的基因产物可能导致新的治疗治疗的潜在靶点。(2)目标1中确定的候选功能的受损感染。将使用单一突变体感染在体内重新检查鉴定的功能。同时，将在临床伤口分离株中灭活候选基因，以检测在临床菌株背景下是否仍能观察到减毒感染性。(3)对候选基因进行功能表征，以帮助理解减弱感染性的分子基础。试验将测试已知与感染有关的功能是否在突变体中受损，包括生物膜形成、运动性和对吞噬细胞杀伤的抵抗力。该项目的预期成果是全面鉴定铜绿假单胞菌感染所需的基因，并验证糖尿病慢性伤口感染治疗的潜在靶点。