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

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

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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.

项目摘要 Acanthamoeba Castellanii和Naegleria Fowleri是自由生活的变形虫（FLA），导致不可治疗，致命中枢神经系统（CNS）的感染。 FLA是细菌性的，一种环境细菌猎物是铜绿假单胞菌。由于铜绿假单胞菌的进化压力反对大约放牧及其生产大量抗菌化合物的众所周知的能力，我们假设铜绿假单胞菌可能会产生一种或多种分泌的敏锐化合物被识别并充当新的和急需的药物来治疗人类中的变形虫感染。我们已经证明了铜绿假单胞菌的实验室应变的无细胞上清液包含稳定的小对A. castellanii致死的分子。这些化合物将通过验证的管道隔离有机提取，然后是色谱和生物测定指导的分馏。然后他们会使用质谱和NMR表征，以及负责复合生产的基因通过使用生物合成基因簇挖掘工具来识别。分离的化合物将进行毒性测试针对FLA和人类细胞，以及在人类神经元的FLA感染的体外模型中的功效。分别地，我们将通过筛选铜绿假单胞菌转座子来识别杀死变形虫所需的细菌基因突变文库用于脱尾活性。每个突变体的过滤上清液将与A. castellanii孵育和变形虫的生存能力将被量化。我们已经优化了比色测定法，以测量使用磺胺胺B（SRB）的可扩展方式。我们的目标是确定新型治疗剂的潜在客户治疗A. Castellanii和N. Fowleri的毁灭性人类感染。