Role of PA4878 in biofilm antimicrobial resistance

项目说明 铜绿假单胞菌是囊性纤维化(CF)肺部感染的主要病原体之一。 慢性和烧伤伤口。铜绿假单胞菌生物被膜一旦建立，很难用抗菌剂根除。 治疗。生物被膜耐药的性质被认为是多因素的。然而，最近我们在我们的研究中发现 实验室对当前的教条提出了挑战，转而建议存在一种经典的、特定于生物膜的 铜绿假单胞菌的耐药机制。我们的数据表明，生物膜的耐药性是由生物膜调节的- 特异转录调控因子BrlA(PA4878)，MERR转录调节因子家族成员 其在转运体底物结合时激活多个药物转运体的表达。的目标是 拟开展的研究旨在确定BrlA调节P.P.耐药性的机制。 铜绿假单胞菌生物膜。在实验上，我们将首先确定为什么brlA只在生物膜生长中表达 条件。我们假设细菌的物理化学梯度和生长条件 受制于生物膜，负责激活brlA基因的表达。我们将利用brlA-Report 基因融合并将浮游生物生长的报告菌株暴露在“生物膜状”的生长条件下。诱导 BrlA基因的表达将通过荧光进行监测。此外，转座子诱变将被用于 确定在浮游或生物膜生长条件下分别抑制/激活brlA的蛋白质。基于 我们的初步数据，我们假设BrlA是多药外排泵的全球转录调节因子 基因(mexAB-oprM、mexGHI-opmD、mexEF-oprN和oprH-PhoPQ)。以确定BrlA是否充当 转录激活剂，并与brlA、oprH、Mexe、Phop和MexA启动子结合，凝胶迁移率移动和 DNase I足迹分析将用于特定的目的2.BrlA结合的基因/启动子 确认将进一步分析生物膜和类生物膜条件下BrlA依赖的基因表达 使用绿色荧光蛋白报告基因融合。根据我们的初步数据，我们预计将分析 MexAB-oprM和mexEF-oprN。建立BrlA与多药外排泵基因表达之间的紧密联系 基因和生物被膜耐药性、菌落计数和生物被膜MIC测试将在特定目标3至 测定mexAB-oprM和mexEF-oprN等基因突变株的生物被膜耐药性。以防没有墨西哥人 突变株和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突变生物膜(敏感)和MEX突变生物膜显示 中间抗性，并鉴定在感病/抗病中差异产生的膜蛋白 可能导致生物被膜耐药性的生物被膜。这项详细调查的结果预计将 阐明BrlA依赖调控铜绿假单胞菌生物被膜耐药并最终导致铅的机制 以抑制或调节生物膜耐药性为基础的创新和更有效的治疗策略 治疗和控制生物被膜感染。

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.