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

铜绿假单胞菌聚集和分散机制的新型调节剂

基本信息

批准号：
9303254
负责人：
BARBARA I KAZMIERCZAK
金额：
$ 25.13万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-24 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9303254
关键词：
Adhesions Antibiotic Resistance Antibiotics Area Attention Bacteria Bacterial Adhesins Bacterial Infections Behavior Binding Binding Proteins Cells Chronic Communities Complex Cyclic Nucleotides Data Development Devices Ecology Enzymes Exhibits Extracellular Matrix Genes Growth Health Benefit Human Immune In Vitro Industrial Health Industry Infection Intervention Libraries Liquid substance Lung Maps Mediating Medical Medical Device Medicine Microbial Biofilms Multi-Drug Resistance Nosocomial Infections Periodicity Phenotype Play Production Prosthesis Proteins Pseudomonas aeruginosa Regulation Role Second Messenger Systems Signal Transduction Site Structure Surface System Testing Tissues Validation base cell motility cost cystic fibrosis airway cystic fibrosis patients experimental study falls fitness gain of function gene product genetic approach in vivo loss of function mutation microbicide mutant novel pathogen premature prevent promoter receptor response screening

项目摘要

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.

铜绿假单胞菌是一种革兰氏阴性的机会性人体病原体，感染了多个组织站点和医疗设备。铜绿假单胞菌形成强大的生物膜，暴露了高水平的抗性抗生素很难清除。在此应用中，我们建议测试PILZ如何调节生物膜形成和分散。当[C-DI-GMP]水平升高时，缺乏PILZ的细菌迅速聚集在液体中，提出PILZ在传感器中的作用或介导第二个Messenger信号，而C-DI-GMP PILZ的结合基序是退化的，蛋白质不在体外结合环状-D-GMP。在目标1中该建议我们将操纵PILZ的C-DI-GMP结合表面，以确定PILZ是否与体内的C-DI-GMP相互作用。我们已经设计了一种巧妙的基于InSeq的策略来识别基因快速聚类表型所需的产品，并提供原则证明数据，表明筛选策略可以识别出罕见的突变体。最后，流细胞实验将更好地表征PILZ生物膜的主动性，成熟和分散，并指示是否破坏 PILZ功能可以是破坏生物膜种植细菌适应性的策略。