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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：
10084272
负责人：
BARBARA I KAZMIERCZAK
金额：
$ 25.03万
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 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10084272
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 Fractionation Gene Cluster Genes Genetic Genetic Screening Goals 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 granulomatous amebic encephalitis high throughput screening human disease in vitro Model knockout gene macrophage microbial microorganism mortality mutant novel therapeutics opportunistic 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.

项目总结