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: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=646582
• 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.**

大多数细菌作为表面相关的生物膜生长，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，促使人们寻找调节其形成的方法。在寻找生物膜抑制剂的同时，我们鉴定了在亚抑制浓度下刺激铜绿假单胞菌生物膜形成的多种分子。我们推测，生物膜刺激有毒分子是一种防御反应。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亚抑制水平的有害分子。不同抗生素、清洁剂甚至一些抗抑郁药、止吐药和抗癌药物的亚抑制浓度刺激许多细菌物种的生物膜形成。是否每一个都引起了一个独特的反应-或者如果它们聚集在一个共同的损伤或压力反应-是未知的。研究生将：a）将有序的铜绿假单胞菌PA 14转座子突变体文库作为生物膜与刺激分子一起生长，以鉴定不能响应以揭示所涉及的途径的突变体; B）将铜绿假单胞菌lux启动子报告基因文库作为生物膜与/不与刺激物一起生长，以鉴定活性被改变的那些启动子;和c）对用/不用亚抑制浓度的选择刺激物生长的生物膜进行转录谱分析（RNAseq），以比较基因表达模式。选择被生物膜刺激物可靠上调的启动子亚群将作为工具进一步开发（目标2）。*** *2）使用生物膜刺激表型来揭示新的生理学和化学。根据目标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偶联的转录报告基因。这些工具将用于询问B）与刺激反应相比，在给定分子的什么浓度下，我们看到启动子激活？对刺激物（以及它们造成的损害）具有抗性的细菌是否会以与敏感菌株相同的方式作出反应？超生物膜形成者的突变体是否对感兴趣的途径“预激活”？我们还将在一个互补的ORF资助项目“解决抗生素耐药性危机”中利用我们经过验证的报告基因，以发现复杂混合物中亚IC处存在的新分子。重要性：这项工作将揭示细菌检测和响应有毒分子的基本机制，增加生物膜形成，一种常见的表型，并将指出新的和更好的方法来操纵生物膜的发展。这些结果将为可能需要增加生物膜形成的场景提供信息-例如废水处理，从尾矿中提取矿物质，益生菌和益生菌的开发，或植物根部定植以促进生长。