Role of PA4878 in biofilm antimicrobial resistance

PA4878 在生物膜抗菌素耐药性中的作用

• 批准号: 8312491
• 负责人: Karin Sauer
• 金额: $ 25.88万
• 依托单位: STATE UNIVERSITY OF NY,BINGHAMTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8312491
• 关键词: Antibiotic Resistance; Antibiotics; Antimicrobial Resistance; Bacteria; Binding; Biological Assay; Burn injury; Cause of Death; Cells; Chronic; Data; Family; Fluorescence; Gel; Gene Expression; Gene Expression Regulation; Gene Fusion; Genes; Goals; Growth; Hydrogen Peroxide; In Vitro; Infection; Intermediate resistance; Investigation; Laboratories; LacZ Genes; Lead; Link; Membrane Proteins; Microbial Biofilms; Monitor; Mutagenesis; Nature; Nosocomial Infections; Phase; Phenotype; Proteins; Pseudomonas aeruginosa; Publishing; Pulmonary Cystic Fibrosis; Regulation; Reporter; Reporter Genes; Research; Resistance; Role; Testing; Time; Transcription Coactivator; antimicrobial; antimicrobial drug; base; cystic fibrosis patients; effective therapy; efflux pump; in vivo; innovation; member; multi drug transporter; mutant; novel; overexpression; pathogen; promoter; public health relevance; resistance mechanism; treatment strategy

PROJECT DESCRIPTION P. aeruginosa is one of the principal pathogens associated with Cystic fibrosis (CF) pulmonary infection and chronic and burn wounds. Once established, P. aeruginosa biofilms are difficult to eradicate by antimicrobial treatment. The nature of biofilm resistance has been deemed multifactorial. However, recent findings in our laboratory challenge the current dogma and suggest instead the existence of a classical, biofilm-specific mechanism of resistance in P. aeruginosa. Our data suggest that biofilm resistance is regulated by the biofilm- specific transcriptional regulator BrlA (PA4878), a member of the MerR family of transcriptional regulators which activate expression of multi drug transporters upon binding of the transporter substrate. The goal of the proposed study is to determine the mechanism by which BrlA regulates antimicrobial resistance of P. aeruginosa biofilms. Experimentally, we will first determine why brlA is only expressed under biofilm growth conditions. We hypothesize that physicochemical gradients and growth conditions to which bacteria are subjected to in biofilms, are responsible for activating brlA gene expression. We will make use of brlA-reporter gene fusions and expose reporter strains grown planktonically to "biofilm-like" growth condition. Induction of brlA gene expression will be monitored via fluorescence. Furthermore, transposon mutagenesis will be used to identify proteins that repress/activate brlA under planktonic or biofilm growth conditions, respectively. Based on our preliminary data, we hypothesize that BrlA is a global transcriptional regulator of multidrug efflux pump genes (mexAB-oprM, mexGHI-opmD, mexEF-oprN, and oprH-phoPQ). To determine whether BrlA acts as transcriptional activator and binds to the promoters of brlA, oprH, mexE, phoP, and mexA, gel mobility shift and DNAse I footprinting assays will be used in Specific Aim 2. Genes/promoters for which BrlA-binding has been confirmed will be further analyzed for BrlA-dependent gene expression in biofilms and biofilm-like condition using gfp reporter gene fusions. Based on our preliminary data, we anticipate analyzing the expression of mexAB-oprM and mexEF-oprN. To establish a firm link between BrlA, expression of multidrug efflux pump genes, and biofilm resistance, CFU counts and biofilm MIC testing will be carried out in Specific Aim 3 to determine biofilm resistance of isogenic mutants of mexAB-oprM and mexEF-oprN. In case none of the mex mutants are as susceptible as brlA biofilms, we will quantitatively compare the membrane protein composition of biofilms by P. aeruginosa PAO1, brlA mutant biofilms (susceptible), and mex mutant biofilms showing intermediate resistance, and identify membrane proteins that are differentially produced in susceptible/resistant biofilms that may contribute to biofilm resistance. Findings from this detailed investigation are expected to elucidate the mechanism of BrlA-dependent regulation of P. aeruginosa biofilm resistance and eventually lead to innovative and more effective treatment strategies based on inhibition or regulation of biofilm resistance to treat and control biofilm infections.

项目描述 铜绿假单胞菌是与囊性纤维化（CF）肺部感染相关的主要病原体之一 慢性和烧伤伤口。建立后，铜绿假单胞菌生物膜很难通过抗菌剂根除 治疗。生物膜耐药性的性质被认为是多因素的。但是，我们的最新发现 实验室挑战当前的教条，而是建议存在经典的生物膜特异性 铜绿假单胞菌的抗性机制。我们的数据表明，生物膜耐药性受到生物膜的调节 特定的转录调节器BRLA（PA4878），MERR转录调节器家族的成员 在转运蛋白底物结合后，激活多药转运蛋白的表达。目标的目标 拟议的研究是确定BRLA调节P的抗菌抗性的机制。 铜绿生物膜。在实验上，我们将首先确定为什么仅在生物膜生长下表达BRLA 状况。我们假设细菌是物理化学梯度和生长条件 受到生物膜的影响，负责激活BRLA基因表达。我们将利用Brla-Reporter 基因融合并暴露了记者菌株，浮游生物在“生物膜样”的生长状态下生长。诱导 BRLA基因表达将通过荧光监测。此外，转座子诱变将用于 分别鉴定在浮游或生物膜生长条件下抑制/激活BRLA的蛋白质。基于 我们的初步数据，我们假设BRLA是多饮用外泵的全球转录调节器 基因（mexab-oprm，mexghi-opmd，mexef-oprn和oprh-phopq）。确定BRLA是否充当 转录激活剂并与BRLA，OPRH，MEXE，PHOP和MEXA的启动子结合，凝胶迁移率和 DNase I足迹测定将用于特定的AIM 2。 确认将进一步分析生物膜中BRLA依赖性基因表达和生物膜样条件 使用GFP报告基因融合。根据我们的初步数据，我们预计分析的表达 mexab-oprm和mexef-oprn。为了在BRLA之间建立牢固的联系，表达了多饮用外泵 基因和生物膜耐药性，CFU计数和生物膜MIC测试将在特定的目标3中进行 确定Mexab-Oprm和Mexef-oprn的亚源性突变体的生物膜抗性。万一没有一个 突变体与BRLA生物膜一样敏感，我们将定量比较膜蛋白组成 铜绿假单胞菌PAO1，BRLA突变体生物膜（易感）和MEX突变体生物膜的生物膜生物膜 中等电阻，并鉴定在易感/抗性中差异产生的膜蛋白 可能导致生物膜耐药性的生物膜。预计此详细调查的结果将 阐明铜绿假单胞菌生物膜抗性的BRLA依赖性调节机制，并最终导致 基于抑制或调节生物膜抵抗的创新和更有效的治疗策略 治疗和控制生物膜感染。