Role of PA4878 in biofilm antimicrobial resistance

项目描述 P.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。一旦建立，铜绿假单胞菌生物膜难以通过抗菌剂根除。 治疗生物膜抗性的性质被认为是多因素的。然而，我们最近的研究结果表明， 实验室挑战目前的教条，并建议，而不是存在一个经典的，生物膜特异性 铜绿假单胞菌的耐药机制。我们的数据表明生物膜的抗性是由生物膜调节的- 特异性转录调节因子BrlA（PA 4878），转录调节因子MerR家族的成员 其在结合转运蛋白底物时激活多药物转运蛋白的表达。的目标 拟开展的研究旨在确定BrlA调节P. 铜绿生物膜实验上，我们将首先确定为什么brlA仅在生物膜生长下表达 条件我们假设细菌生长所需的物理化学梯度和生长条件 在生物膜中，负责激活brlA基因表达。我们将利用brlA报告员 基因融合，并将报道菌株暴露于“生物膜样”生长条件下。诱导 通过荧光监测brlA基因表达。此外，转座子诱变将用于 鉴定分别在促生长或生物膜生长条件下抑制/激活brlA的蛋白质。基于 根据我们的初步数据，我们假设BrlA是多药外排泵的全局转录调节因子， 基因（mexAB-oprM、mexGHI-opmD、mexEF-oprN和oprH-phoPQ）。为了确定BrlA是否作为 转录激活因子并结合brlA、oprH、mexE、phoP和mexA的启动子，凝胶迁移率改变和 DNA酶I足迹分析将用于特异性目的2。BrlA结合已经被抑制的基因/启动子 将进一步分析生物膜和生物膜样条件下BrlA依赖性基因表达 使用GFP报告基因融合体。根据我们的初步数据，我们预计分析的表达 mexAB-oprM和mexEF-oprN。建立BrlA、多药外排泵表达和细胞凋亡之间的紧密联系 基因，生物膜抗性，CFU计数和生物膜MIC检测将在特定目标3中进行， 测定mexAB-oprM和mexEF-oprN的同基因突变体的生物膜抗性。如果没有一个 Mex 突变体就像 brlA生物膜，我们将定量比较膜蛋白组成 铜绿假单胞菌PAO 1的生物膜， brlA突变体生物膜（易感的），和 mex突变生物膜显示 中间抗性，并鉴定在易感/抗性中差异产生的膜蛋白 这可能有助于生物膜抗性。这项详细调查的结果预计将 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 本发明涉及基于抑制或调节生物膜抵抗性的创新和更有效的治疗策略， 治疗和控制生物膜感染。

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.