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

项目摘要 卡氏棘阿米巴和福氏奈格勒阿米巴是一种自由生活的阿米巴(FLA)，可导致无法治疗的、致命的 中枢神经系统(CNS)感染。FLA是食菌性的，他们有一种环境细菌 猎物是铜绿假单胞菌。因为铜绿假单胞菌自我防御的进化压力 反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能产生一种或多种分泌的阿米巴杀菌化合物，这种化合物可以 确定并作为治疗人类阿米巴感染的新的和急需的药物的线索。 我们已经证明，实验室菌株的无细胞培养上清液中含有稳定的小分子 对卡氏曲霉有致死作用的分子(S)。这些化合物(S)将通过一条成熟的管道进行分离 有机提取，然后层析和生物测定引导的分级。到那时，他们将成为 用质谱学和核磁共振进行了表征，负责化合物生产的基因将是 利用生物合成基因簇挖掘工具进行鉴定。分离出的化合物(S)将进行毒性测试 抗FLA和人类细胞，以及在人类神经元FLA感染的体外模型中的疗效。另外， 我们将通过筛选排列的铜绿假单胞菌转座子来鉴定杀死阿米巴所需的细菌基因。 阿米巴杀灭活性突变体库。每个突变体的过滤上清液将与卡氏拟青霉培养。 阿米巴的生存能力将被量化。我们已经优化了一种比色法来测量阿米巴在体内的活性 使用磺胺类胡丹明B(SRB)的可扩展方式。我们的目标是为新的治疗药物寻找线索 治疗由卡氏拟青霉和福勒氏乳杆菌引起的毁灭性的人类感染。

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.