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（由申请人提供）：铜绿假单胞菌是与囊性纤维化（CF）肺部感染以及慢性和烧伤伤口相关的主要病原体之一。一旦建立，铜绿假单胞菌生物膜就难以通过抗菌治疗消除。生物膜耐药性的性质被认为是多因素的。但是，我们实验室中的最新发现挑战了当前的教条，而是暗示存在铜绿假单胞菌中经典的，生物膜特异性的机制。我们的数据表明，生物膜耐药性受生物膜特异性转录调节剂BRLA（PA4878）的调节，这是转录调节剂MERR家族的成员，该家族在转运蛋白底物结合后激活多药物转运蛋白的表达。拟议的研究的目的是确定BRLA调节铜绿假单胞菌生物膜抗菌抗性的机制。在实验上，我们将首先确定为什么仅在生物膜生长条件下表达BRLA。我们假设细菌在生物膜中受到的物理化学梯度和生长条件负责激活BRLA基因表达。我们将利用BRLA-REPORTER基因融合，并暴露于浮游生物中的报告菌株，以“生物膜样”的生长条件。 BRLA基因表达的诱导将通过荧光监测。此外，转座子诱变将用于鉴定分别在浮游生物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是多饮出外排泵基因（Mexab-oprm，Mexghi-opMD，Mexef-oprn和oprh-phopq）的全球转录调节剂。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，将在特定的AIM/启动子中使用BRLA结合的基因/促进剂在特定的AIM/启动子中使用BRLA结合的特定AIM/启动子，将进一步分析BRLA依赖性的基因和依赖性基因的基因/促进剂，并将其用于依赖BRLA的表达。记者基因融合。根据我们的初步数据，我们预计分析Mexab-Oprm和Mexef-oprn的表达。为了在BRLA之间建立牢固的联系，将在特定的目标3中进行多种外排泵基因的表达以及生物膜耐药性，CFU计数和生物膜MIC测试，以确定MEXAB-OPRM和MEXEF-OPRN的异源性突变体的生物膜耐药性。如果没有任何MEX突变体像BRLA生物膜那样易感，我们将通过铜绿假单胞菌PAO1，BRLA突变体生物膜（易感）（易感）（易感）和MEX突变体生物膜和MEX突变体的生物蛋白具有抗性抗性，并确定膜蛋白具有不同的生物蛋白，并确定膜蛋白具有不同的效果，并确定膜蛋白具有抗性，并确定膜蛋白具有不同的效果，并确定膜蛋白具有不同的效果，并确定膜蛋白具有不同生物膜耐药性。这项详细研究的结果有望阐明BRLA依赖性铜绿假单胞菌生物膜耐药性的机制，并最终基于抑制或调节生物膜耐药性来治疗和控制生物膜感染的创新和更有效的治疗策略。公共卫生相关性：铜绿假单胞菌是囊性纤维化患者的医院后感染和主要死亡原因的常见原因。铜绿假单胞菌的标志之一是其对抗生素的固有耐药性。该建议旨在了解生物膜耐药性的机理以及新型转录调节剂BRLA在调节铜绿假单胞菌生物膜的抗菌抗性中的作用。这项研究的发现可能会导致新颖和创新的治疗策略，以治疗和消除生物膜感染。