Agents Provocateur: Exploiting bacterial biofilm stimulation to identify bioactive small molecules

Agents Provocateur：利用细菌生物膜刺激来识别生物活性小分子

• 批准号: RGPIN-2016-06521
• 负责人: Burrows, Lori
• 金额: $ 2.77万
• 依托单位: McMaster University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687058
• 关键词: Agents; Provocateur; Exploiting; bacterial; biofilm

Most bacteria grow as surface-associated biofilms, with reduced susceptibility to antibiotics and disinfectants. Biofilms are important in industrial, agricultural, and medical settings, prompting searches for ways to modulate their formation. While searching for biofilm inhibitors, we identified diverse molecules that stimulated Pseudomonas aeruginosa biofilm formation at sub-inhibitory concentrations. We hypothesize that biofilm stimulation by toxic molecules is a defensive response. We will pursue two objectives:***1) Determine how P. aeruginosa perceives and responds to sub-inhibitory levels of noxious molecules. Sub-inhibitory concentrations of diverse antibiotics, detergents, and even some antidepressants, anti-emetics, and anti-cancer drugs stimulate biofilm formation of many bacterial species. Whether each evokes a unique response or if they converge on a common damage or stress response is unknown. A graduate student will: a) grow an ordered P. aeruginosa PA14 transposon mutant library as biofilms with stimulatory molecules to identify mutants unable to respond to uncover the pathways involved; b) grow a P. aeruginosa lux promoter reporter library as biofilms with/without stimulators to identify those promoters whose activity is altered; and c) perform transcriptional profiling (RNAseq) of biofilms grown with/without sub-inhibitory concentrations of select stimulators, to compare patterns of gene expression. Select promoter subsets that are reliably up-regulated by biofilm stimulators will be further developed as tools (Obj. 2).*** ***2) Use the biofilm-stimulation phenotype to uncover new physiology and chemistry. Informed by the pathways identified in Obj 1, a postdoctoral fellow will a) develop and validate transcriptional reporters coupled to promoters that are activated by biofilm-stimulatory molecules. These tools will be used to ask b) at what concentration of a given molecule do we see promoter activation compared with the stimulation response? Do bacteria that are resistant to stimulators (and the damage they cause) respond in the same way as a susceptible strain? Are mutants that are hyper-biofilm formers pre-activated' for the pathways of interest? We will also leverage our validated reporters in a complementary ORF-funded project, Solving the antibiotic resistance crisis', to uncover new molecules present at sub-IC in complex mixtures.***Significance: This work will uncover the basic mechanisms by which bacteria detect and respond to toxic molecules with increased biofilm formation, a common phenotype, and will point to new and better ways to manipulate biofilm development. The results will prove informative for scenarios in which increased biofilm formation may be desirable e.g. in wastewater treatment, extraction of minerals from mine tailings, development of pre- and probiotics, or plant root colonization for growth promotion.**

大多数细菌以表面相关生物膜的形式生长，对抗生素和消毒剂的敏感性降低。生物膜在工业、农业和医疗环境中很重要，促使人们寻找方法来调节它们的形成。在寻找生物被膜抑制剂的同时，我们确定了在亚抑制浓度下刺激铜绿假单胞菌生物被膜形成的不同分子。我们假设有毒分子对生物膜的刺激是一种防御性反应。我们将追求两个目标：*1)确定铜绿假单胞菌如何感知和响应有毒分子的亚抑制水平。不同的抗生素、洗涤剂，甚至一些抗抑郁剂、止吐和抗癌药物的亚抑制浓度会刺激许多细菌物种的生物被膜形成。每种反应是否会引起一种独特的反应，或者它们是否会聚在一种共同的损伤或压力反应上，目前尚不清楚。研究生将：a)培养有序的铜绿假单胞菌PA14转座子突变体文库，作为带有刺激分子的生物膜，以识别无法反应以揭示相关途径的突变体；b)培养铜绿假单胞菌lux启动子报告文库，作为有/无刺激子的生物膜，以鉴定其活性发生改变的启动子；以及c)对有/无亚抑制浓度的选定刺激子的生物膜进行转录谱分析(RNAseq)，以比较基因表达的模式。被生物膜刺激物可靠上调的部分启动子亚群将作为工具进一步开发(Obj.2)利用生物膜刺激表型发现新的生理和化学特性。根据OBJ 1中确定的途径，博士后研究员将a)开发和验证与生物膜刺激分子激活的启动子耦合的转录报告。这些工具将被用来问b)在给定的分子浓度下，与刺激反应相比，我们看到启动子激活了什么？对刺激物(及其造成的损害)具有抵抗力的细菌是否会以与敏感菌株相同的方式做出反应？作为超生物膜形成者的突变体是否为感兴趣的途径预先激活？我们还将利用我们经过验证的记者在一个由ORF资助的补充项目中，解决抗生素耐药性危机，以发现复杂混合物中亚IC存在的新分子。*意义：这项工作将揭示细菌检测和响应有毒分子的基本机制，增加生物膜的形成，这是一种常见的表型，并将指出操纵生物膜发展的新的和更好的方法。结果将证明，在废水处理、从尾矿中提取矿物质、开发前和益生菌的开发，或促进生长的植物根部定植等方面，可能需要增加生物膜形成的情况下，这将是有益的。