Role of PA4878 in biofilm antimicrobial resistance

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、慢性和烧伤伤口相关的主要病原体之一。一旦形成，铜绿假单胞菌生物膜很难通过抗菌治疗根除。生物膜耐药性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，并建议铜绿假单胞菌存在经典的，生物膜特异性的耐药机制。我们的数据表明，生物膜耐药性是由生物膜特异性转录调节剂BrlA （PA4878）调控的，BrlA是MerR转录调节剂家族的一员，在与转运体底物结合时激活多种药物转运体的表达。本研究的目的是确定BrlA调控铜绿假单胞菌生物膜耐药性的机制。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受到的物理化学梯度和生长条件是激活brlA基因表达的原因。我们将利用brla报告基因融合，并将浮游生长的报告菌株暴露在“生物膜样”生长条件下。通过荧光监测brlA基因表达的诱导情况。此外，转座子诱变将分别用于在浮游或生物膜生长条件下鉴定抑制/激活brlA的蛋白质。根据我们的初步数据，我们假设BrlA是多药物外排泵基因（mexAB-oprM, mexGHI-opmD， mexEF-oprN和oprH-phoPQ）的全球转录调节因子。为了确定BrlA是否作为转录激活剂并与BrlA、oprH、mexE、phoP和mexA的启动子结合，将在Specific Aim 2中使用凝胶迁移转移和DNAse I足迹测定。已确认与brla结合的基因/启动子将使用gfp报告基因融合进一步分析brla依赖基因在生物膜和生物膜样条件下的表达。基于我们的初步数据，我们期待分析mexAB-oprM和mexEF-oprN的表达。为了建立BrlA、多药外排泵基因表达与生物膜耐药之间的紧密联系，将在Specific Aim 3中进行CFU计数和生物膜MIC检测，以确定mexAB-oprM和mexEF-oprN等基因突变体的生物膜耐药。如果没有一个mex突变体像brlA生物膜一样敏感，我们将定量比较P. aeruginosa PAO1、brlA突变体生物膜（易感）和mex突变体生物膜的膜蛋白组成，并确定易感/耐药生物膜中不同产生的膜蛋白，这些膜蛋白可能有助于生物膜的抗性。这项详细的研究结果有望阐明brla依赖性调节铜绿假单胞菌生物膜耐药性的机制，并最终导致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其对抗生素的高内在耐药性。本研究旨在了解P. aeruginosa生物膜耐药机制，以及新型转录调控因子BrlA在调控P. aeruginosa生物膜耐药中的作用。这项研究的发现可能会导致新的和创新的治疗策略来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.