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

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，促使人们寻找调节其形成的方法。在寻找生物膜抑制剂的过程中，我们发现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。我们假设有毒分子对生物膜的刺激是一种防御反应。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。各种抗生素、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。是否每一种都会引起一种独特的反应，或者它们是否会聚成一种共同的损伤或应激反应，目前还不得而知。研究生将：A)培养一个有序的铜绿假单胞菌PA14转座子突变文库，作为带有刺激分子的生物膜，以识别无法响应的突变体，从而揭示所涉及的途径；b)培养铜绿假单胞菌光通量启动子报告文库作为生物膜，以识别活性改变的启动子；c)对有/没有亚抑制浓度的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。选择被生物膜刺激剂可靠上调的启动子亚群将作为进一步开发的工具（Obj. 2）.*** ***2)利用生物膜刺激表型揭示新的生理和化学。根据Obj 1中确定的途径，博士后研究员将a)开发并验证与启动子偶联的转录报告子，这些启动子被生物膜刺激分子激活。这些工具将用于问b)在给定分子的什么浓度下，与刺激反应相比，我们看到启动子激活？对刺激物（以及刺激物造成的损害）有抵抗力的细菌与敏感菌株的反应方式相同吗？超生物膜形成的突变体是否对感兴趣的途径“预激活”？我们还将在orf资助的补充项目“解决抗生素耐药性危机”中利用我们经过验证的报告者，发现复杂混合物中亚ic中存在的新分子。***意义：这项工作将揭示细菌检测和响应有毒分子增加生物膜形成的基本机制，这是一种常见的表型，并将指出新的和更好的方法来操纵生物膜的形成。该结果将证明在某些情况下可能需要增加生物膜的形成-例如在废水处理，从矿山尾矿中提取矿物质，开发预益生菌和益生菌，或植物根定殖以促进生长

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.**