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

铜绿假单胞菌产生的致病性自由生活阿米巴杀阿米巴产品的鉴定

基本信息

批准号：
9885697
负责人：
BARBARA I KAZMIERCZAK
金额：
$ 20.85万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-10 至 2021-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9885697
关键词：
Acanthamoeba castellanii Amoeba genus Bacteria Bacterial Genes Biological Assay Brain Cells Central Nervous System Infections Chemicals Chromatography Collaborations Complement Complex Data Drug Design Ecology Encephalitis Fractionation Gene Cluster Genes Genetic Genetic Screening Goals Granulomatous Human Incubated Infection Laboratories Lethal Dose 50 Libraries Liquid Chromatography Mass Spectrum Analysis Measures Mining Molecular Naegleria fowleri Natural Product Drug Natural Products Neuraxis Neurons Pathogenesis Pathogenicity Pathway interactions Patients Pharmaceutical Preparations Phenotype Predatory Behavior Production Pseudomonas Pseudomonas aeruginosa Research Signal Transduction Soil Source Specificity Spinal Cord Structure Survivors Therapeutic Agents Toxic effect Toxicity Tests Water Work antimicrobial cell type drug candidate drug discovery effective therapy high throughput screening human disease in vitro Model knockout gene macrophage microbial microorganism mortality mutant novel therapeutics pathogen pressure primary amebic meningoencephalitis screening small molecule tool

项目摘要

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.

项目概要卡氏棘阿米巴和福氏耐格里阿米巴是自由生活的阿米巴 (FLA)，会导致无法治愈的致命疾病中枢神经系统（CNS）感染。 FLA 是食菌性的，它们是一种环境细菌其捕食对象是铜绿假单胞菌。由于铜绿假单胞菌保护自身的进化压力针对变形虫的放牧及其众所周知的产生大量抗菌化合物的能力，我们假设铜绿假单胞菌可能产生一种或多种分泌的杀阿米巴化合物确定并作为治疗人类阿米巴感染的新的和急需的药物的先导。我们已经证明，铜绿假单胞菌实验室菌株的无细胞上清液含有稳定的小分子对 A.castellanii 致命的分子。这些化合物将通过经过验证的管道进行分离有机萃取，然后进行色谱法和生物测定引导的分级分离。那时他们将是使用质谱和核磁共振进行表征，负责化合物产生的基因将是通过利用生物合成基因簇挖掘工具来识别。将测试分离出的化合物的毒性针对 FLA 和人类细胞，以及在 FLA 感染人类神经元的体外模型中的功效。分别地，我们将通过筛选排列好的铜绿假单胞菌转座子来鉴定杀死阿米巴原虫所需的细菌基因具有杀阿米巴活性的突变体文库。来自每个突变体的过滤上清液将与 A.castellanii 一起孵育阿米巴原虫的生存能力将被量化。我们优化了比色测定法来测量阿米巴原虫的活力使用磺胺罗丹明 B (SRB) 进行可扩展的方式。我们的目标是确定新型治疗药物的线索治疗由 A.castellanii 和 N.fowleri 引起的毁灭性人类感染。