Identification of Ameobicidal Products Against Pathogenic Free-Living Amoebae Produced by Pseudomonas aeruginosa

项目摘要 卡氏阿米巴和福氏耐格里阿米巴是自由生活的阿米巴（弗拉）， 中枢神经系统（CNS）感染。弗拉是食菌的，一种环境细菌， 是铜绿假单胞菌。因为铜绿假单胞菌为了保护自己 针对阿米巴放牧及其众所周知的产生过多抗菌化合物的能力，我们 假设铜绿假单胞菌可以产生一种或多种分泌的杀阿米巴化合物， 确定并作为新的和急需的药物，以治疗人类阿米巴感染的领导。 我们已经证明，实验室铜绿假单胞菌菌株的无细胞上清液含有稳定的小分子。 对A. castellanii。这些化合物将通过一个经过验证的管道分离， 有机提取，然后进行色谱法和生物测定指导的分馏。于其后向业主悉数 使用质谱和NMR表征，负责化合物产生的基因将被 通过利用生物合成基因簇挖掘工具识别。将对分离的化合物进行毒性试验 针对弗拉和人细胞的有效性以及在弗拉感染人神经元的体外模型中的有效性。另外， 我们将通过筛选排列的铜绿假单胞菌转座子， 杀阿米巴活性的突变体文库。将各突变体的过滤上清液与A.卡斯特拉尼 并对变形虫的生存力进行定量。我们优化了一种比色测定法，以测量变形虫的活力， 使用磺酰罗丹明B（SR B）的可扩展方式。我们的目标是确定新的治疗药物的线索， 治疗毁灭性的人类感染。castellanii和N.福勒里

Project Summary Acanthamoeba castellanii and Naegleria fowleri are free-living amoebae (FLA) that cause untreatable, fatal infections of the central nervous system (CNS). FLA are bacterivorous, and one environmental bacterium they prey upon is Pseudomonas aeruginosa. Because of the evolutionary pressure for P. aeruginosa to defend itself against amoebal grazing and its well-known ability to produce a plethora of antimicrobial compounds, we hypothesize that P. aeruginosa may produce one or more secreted amoebicidal compounds that can be identified and serve as leads for new and much-needed drugs to treat amoeba infections in humans. We have demonstrated that the cell-free supernatants of a laboratory strain of P. aeruginosa contain stable small molecule(s) that are lethal to A. castellanii. These compound(s) will be isolated through a proven pipeline of organic extraction followed by chromatography and bioassay-guided fractionation. They will then be characterized using mass spectrometry and NMR, and the genes responsible for compound production will be identified by utilizing biosynthetic gene cluster mining tools. The isolated compound(s) will be tested for toxicity against FLA and human cells and for efficacy in an in vitro model of FLA infection of human neurons. Separately, we will identify bacterial genes required to kill amoebae by screening an arrayed P. aeruginosa transposon mutant library for amoebicidal activity. Filtered supernatants from each mutant will be incubated with A. castellanii and amoeba viability will be quantified. We have optimized a colorimetric assay to measure amoeba viability in a scalable manner using sulforhodamine B (SRB). It is our goal to identify leads for novel therapeutic agents to treat devastating human infections by A. castellanii and N. fowleri.