Glutamate-dependent behavior of Pseudomonas aeruginosa

描述（由申请方提供）：铜绿假单胞菌是一种机会致病菌，可引起囊性纤维化（CF）患者、烧伤患者、呼吸机患者和肠道重建患者的急性和持续感染。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，这些感染中最持久的，也是最难用抗生素治疗的，是那些形成细菌生物膜的感染。通常，从无张力状态建立生物膜的第一步是群集，其中细菌利用表面运动性成群地定殖新表面。先前关于铜绿假单胞菌群集的报道已经显示了称为鼠李糖脂的表面活性剂的产生的重要性，但是最近确定当与谷氨酸盐一起生长时，鼠李糖脂的产生对于群集不是必需的。这种谷氨酸依赖性表型与发病机制有明显的联系，特别是对于肺部感染。例如，氨基酸营养缺陷型通常从CF患者的肺痰中分离。 转座子诱变方法将用于确定赋予谷氨酸作用的基因，然后系统地表征和确认显示谷氨酸群集损失的随机突变体的重要性。将使用蛋白质组学、湿化学和分析化学方案探索铜绿假单胞菌在谷氨酸盐上生长时产生的表面活性剂的分子性质、调节和活性，以表征该表面活性剂及其合成所需的蛋白质。将使用生物膜和细胞毒性测定来检查该表面活性剂的更广泛的活性。使用干净的缺失突变体和化学数据，将在包括合成CF痰液在内的各种培养基上探索这种谷氨酸依赖性群体表型的重要性。 了解谷氨酸等氨基酸如何决定铜绿假单胞菌的群体运动和生物膜发育，可能会大大提高我们对发病机制的理解。这些知识对于控制和预防许多呼吸道和医院生物膜感染可能很重要。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性表型的调节反应的新信息，这些表型与铜绿假单胞菌和其他病原体的感染密切相关。这将提高我们对感染开始时细菌行为的理解。此外，由于铜绿假单胞菌的许多毒力活动与阿勒群体感应相关，因此解释这种谷氨酸依赖性表型非常重要，其中阿勒群体感应似乎不需要。

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.