A novel regulator of clumping and dispersal mechanisms in P. aeruginosa

铜绿假单胞菌是一种革兰氏阴性的机会致病菌，可感染多种组织 网站和医疗设备。铜绿假单胞菌形成稳健的生物膜，其表现出高水平的耐药性， 抗生素，很难清除。在本申请中，我们提出测试PilZ如何调节生物膜 形成和扩散。当[c-di-GMP]水平升高时，缺乏PilZ的细菌在液体中迅速聚集， 这表明PilZ在感知或介导第二信使信号中的作用， PilZ的结合基序是简并的，并且该蛋白在体外不结合环-d-GMP。目标1， 本建议我们将操纵PilZ的c-di-GMP结合表面，以确定PilZ是否 与c-di-GMP在体内相互作用。我们设计了一个聪明的基于INSeq的策略来识别基因 快速凝集表型所需的产品，并提供原理证明数据， 该筛选策略可以鉴定不能聚集的罕见突变体。最后，流动池实验将 更好地表征pilZ生物膜的起始、成熟和分散，并指示是否破坏 可以是破坏生物膜生长的细菌的适应性的策略。

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that infects multiple tissue sites and medical devices. P. aeruginosa forms robust biofilms that exhibit high levels of resistance to antibiotics and are difficult to clear. In this application, we propose to test how PilZ regulates biofilm formation and dispersal. Bacteria lacking PilZ rapidly clump in liquid when [c-di-GMP] levels rise, suggesting a role for PilZ in sensing or mediating this second messenger signal—and yet the c-di-GMP binding motifs of PilZ are degenerate, and the protein does not bind cyclic-d-GMP in vitro. In Aim 1 of this proposal we will manipulate the c-di-GMP binding surface of PilZ, to determine whether PilZ interacts with c-di-GMP in vivo. We have devised a clever INSeq based strategy to identify gene products required for the rapid clumping phenotype, and present proof-of-principle data showing that the screening strategy can identify rare mutants that fail to clump. Lastly, flow cell experiments will better characterize initiation, maturation and dispersal of pilZ biofilms, and indicate whether disruption of PilZ function can be a strategy to disrupt fitness of biofilm grown bacteria.