Role of PA4878 in biofilm antimicrobial resistance

描述（由申请方提供）：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微生物处理根除。生物膜抗性的性质被认为是多因素的。然而，我们实验室最近的研究结果挑战了目前的教条，并建议在铜绿假单胞菌中存在经典的生物膜特异性耐药机制。我们的数据表明，生物膜抗性由生物膜特异性转录调节因子BrlA（PA 4878）调节，该转录调节因子是转录调节因子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。所提出的研究的目标是确定BrlA调节铜绿假单胞菌生物膜的抗微生物剂抗性的机制。在实验上，我们将首先确定为什么brlA仅在生物膜生长条件下表达。我们假设，物理化学梯度和生长条件，细菌在生物膜中受到，负责激活brlA基因表达。我们将利用brlA-报告基因融合体，并将非稳态生长的报告菌株暴露于“生物膜样”生长条件。通过荧光监测brlA基因表达的诱导。此外，转座子诱变将用于分别鉴定在植物生长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是多药外排泵基因（mexAB-oprM、mexGHI-opmD、mexEF-oprN和oprH-phoPQ）的全局转录调节因子。为了确定BrlA是否充当转录激活因子并与brlA、oprH、mexE、phoP和mexA的启动子结合，将在特异性目标2中使用凝胶迁移率变动和DNA酶I足迹分析。将使用gfp报告基因融合物进一步分析已经确认BrlA结合的基因/启动子在生物膜和生物膜样条件下的BrlA依赖性基因表达。基于我们的初步数据，我们预期分析mexAB-oprM和mexEF-oprN的表达。为了在BrlA、多药外排泵基因的表达和生物膜抗性之间建立牢固的联系，将在特定目标3中进行CFU计数和生物膜MIC测试，以确定mexAB-oprM和mexEF-oprN的同基因突变体的生物膜抗性。在没有mex突变体像brlA生物膜一样敏感的情况下，我们将定量比较铜绿假单胞菌PAO 1、brlA突变体生物膜（敏感的）和显示中等抗性的mex突变体生物膜的膜蛋白组成，并鉴定在敏感/抗性生物膜中差异产生的可能有助于生物膜抗性的膜蛋白。来自该详细研究的发现预期阐明铜绿假单胞菌生物膜抗性的BrlA依赖性调节的机制，并最终导致基于生物膜抗性的抑制或调节的创新且更有效的治疗策略，以治疗和控制生物膜感染。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。铜绿假单胞菌的特征之一是其对抗生素的高内在抗性。本研究旨在了解铜绿假单胞菌生物膜耐药性的机制以及新型转录调控因子BrlA在调节铜绿假单胞菌生物膜耐药性中的作用。这项研究的结果可能会导致新的和创新的治疗策略，以治疗和根除生物膜感染。

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.