Glutamate-dependent behavior of Pseudomonas aeruginosa

描述(由申请人提供)：铜绿假单胞菌是一种机会性病原体，对囊性纤维化(CF)患者、烧伤患者、呼吸机患者和接受肠道重建的患者的急性和持续性感染都有责任。铜绿假单胞菌是重症监护病房患者最常见的医院病原体之一。对于铜绿假单胞菌和许多细菌病原体来说，这些感染中最持久、最难用抗生素治疗的是那些形成细菌生物膜的感染。通常，从浮游状态建立生物膜的第一步是蜂拥而至，细菌利用表面运动性成群地在新表面定居。以前关于铜绿假单胞菌成群生长的报告表明，生产一种名为鼠李糖脂的表面活性物质很重要，但最近确定，当与谷氨酸一起生长时，生产鼠李糖脂不是成群生长所必需的。这种依赖谷氨酸的表型与发病机制有明显的联系，特别是在肺部感染方面。例如，从CF患者的肺痰中常规分离出氨基酸营养缺乏症细胞。转座子突变方法将被用来确定赋予谷氨酸效应的基因，然后系统地表征和确认表现出对谷氨酸的群集性丧失的随机突变体的重要性。铜绿假单胞菌产生的表面活性物质在谷氨酸上生长时的分子性质、调节和活性将利用蛋白质组学、湿化学和分析化学方法进行探索，以表征这种表面活性物质及其合成所需的蛋白质(S)。这种表面活性剂的更广泛的活性将通过生物被膜和细胞毒性试验来检验。利用干净的缺失突变体和化学数据，这种依赖谷氨酸的群体表型的重要性将在包括合成CF痰在内的各种介质上进行探索。弄清谷氨酸等氨基酸是如何决定铜绿假单胞菌的群体运动和生物膜发育的，可能会极大地提高我们对发病机制的理解。这些知识可能对控制和预防许多呼吸道和医院内生物被膜感染很重要。这项研究的完成将提供有关铜绿假单胞菌对氨基酸、谷氨酸和氨基酸依赖表型的调控反应的新信息，这些表型与铜绿假单胞菌和其他病原体的感染密切相关。这将提高我们对细菌在感染开始时的行为的理解。此外，由于铜绿假单胞菌的许多毒力活动与AHL群体感应相关，解释这种依赖谷氨酸的表型将是重要的，因为AHL群体感应似乎不是必需的。

DESCRIPTION (provided by applicant): Pseudomonas aeruginosa is an opportunistic pathogen responsible for both acute and persistent infections in individuals with cystic fibrosis (CF), burn victims, ventilator patients, and those who have had intestinal reconstruction. P. aeruginosa is among the most common nosocomial pathogens for intensive care unit patients. For P. aeruginosa and many bacterial pathogens, the most persistent of these infections, and the hardest to treat with antibiotics, are those that form bacterial biofilms. Often, a first step n establishing biofilms from the planktonic state is swarming, where bacteria use surface motility to colonize new surfaces in groups. Previous reports for P. aeruginosa swarming have shown importance of production of a surfactant called rhamnolipid, but it was recently determined that rhamnolipid production is not required for swarming when growing with glutamate. Such a glutamate-dependent phenotype has obvious links to pathogenesis, particularly for lung infections. For example, amino acid auxotrophs are routinely isolated from lung sputum of CF patients. A transposon mutagenesis approach will be used to determine the genes that confer the glutamate-effect and then systematically characterize and confirm the importance of the random mutants that show a loss of swarming on glutamate. The molecular nature, regulation, and activity of the surfactant produced by P. aeruginosa when growing on glutamate will be probed using proteomics, wet chemistry, and analytical chemistry protocols to characterize this surfactant and the protein(s) required for its synthesis. The broader activity of this surfactant wll be examined using biofilm and cytotoxicity assays. Using the clean deletion mutants and chemical data, the importance of this glutamate-dependent swarm phenotype will be explored on various media including synthetic CF sputum. Discerning how amino acids like glutamate dictate swarm motility and biofilm development for P. aeruginosa may greatly improve our understanding of pathogenesis. This knowledge may be important to control and prevent many respiratory and nosocomial biofilm infections. Completion of this research will provide new information about a regulatory response of P. aeruginosa to the amino acid glutamate and amino acids-dependent phenotypes that have been strongly associated with infection for P. aeruginosa and other pathogens. This will improve our understanding of bacterial behavior at the onset of infection. Additionally, because many virulence activities of P. aeruginosa are associated with AHL quorum sensing, it will be important to explain this glutamate-dependent phenotype where AHL quorum sensing appears to not be required.