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: 8118963
• 负责人: Karin Sauer
• 金额: $ 29.52万
• 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/8118963
• 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; Health; 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

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a common cause of hospital acquired infection and the leading cause of death in patients with cystic fibrosis. One of the hallmarks of P. aeruginosa is its high intrinsic resistance to antibiotics. This proposal is aimed at understanding the mechanism of biofilm resistance and the role of the novel transcriptional regulator BrlA in regulating antimicrobial resistance of P. aeruginosa biofilms. Findings from this research may lead to novel and innovative treatment strategies to treat and eradicate biofilm infections.

描述(申请人提供)：铜绿假单胞菌是与囊性纤维化(CF)肺部感染、慢性创面和烧伤创面相关的主要病原体之一。一旦形成铜绿假单胞菌生物膜，就很难通过抗菌治疗来根除。生物被膜耐药的性质被认为是多因素的。然而，我们实验室最近的发现挑战了目前的教条，表明铜绿假单胞菌存在一种经典的、生物膜特异性的耐药机制。我们的数据表明，生物膜耐药受到生物膜特异性转录调控因子BrlA(PA4878)的调控，BrlA是MERR转录调节因子家族的成员，它在转运蛋白底物结合时激活多个药物转运蛋白的表达。这项拟议研究的目的是确定BrlA调节铜绿假单胞菌生物被膜耐药性的机制。在实验上，我们将首先确定为什么brlA只在生物膜生长条件下表达。我们假设细菌在生物膜中所经历的物理化学梯度和生长条件负责激活brlA基因的表达。我们将利用brlA报告基因融合，将浮游生物生长的报告菌株暴露在类似生物膜的生长条件下。BrlA基因表达的诱导将通过荧光进行监测。此外，转座子突变将被用来鉴定在浮游或生物膜生长条件下分别抑制/激活brlA的蛋白质。根据我们的初步数据，我们假设BrlA是多药外排泵基因(mexAB-oprM、mexGHI-opmD、mexEF-oprN和oprH-PhoPQ)的全球转录调节因子。为了确定BrlA是否作为转录激活因子并与brlA、oprH、Mexe、Phop和MexA的启动子结合，将在特定的目的2中使用凝胶迁移率漂移和DNase I足迹分析。已确认与BrlA结合的基因/启动子将通过GFP报告基因融合进一步分析BrlA依赖的基因在生物膜和生物膜类条件下的表达。基于我们的初步数据，我们预期分析mexAB-oprM和mexEF-oprN的表达。为了建立BrlA、多药外排泵基因的表达与生物被膜耐药性之间的牢固联系，将针对特定的目标3进行CFU计数和生物被膜MIC测试，以确定mexAB-oprM和mexEF-oprN的同基因突变株的生物被膜耐药性。如果没有一个MEX突变株像brlA生物膜那样敏感，我们将定量比较铜绿假单胞菌PAO1、brlA突变生物膜(敏感)和中等抗性突变MEX生物膜的膜蛋白组成，并鉴定在敏感/耐药生物膜中差异产生的膜蛋白。这项详细研究的结果有望阐明BrlA对铜绿假单胞菌生物被膜耐药性的依赖调节机制，并最终导致基于抑制或调节生物被膜耐药性的创新和更有效的治疗策略，以治疗和控制生物被膜感染。公共卫生相关性：铜绿假单胞菌是医院获得性感染的常见原因，也是囊性纤维化患者死亡的主要原因。铜绿假单胞菌的特点之一是对抗生素具有高度的内在耐药性。本研究旨在了解铜绿假单胞菌生物被膜耐药的机制，以及新型转录调控因子BrlA在铜绿假单胞菌生物被膜耐药调控中的作用。这项研究的发现可能导致治疗和根除生物被膜感染的新颖和创新的治疗策略。