Targeting lipopolysaccharide transport machinery in Pseudomonas aeruginosa

铜绿假单胞菌中的靶向脂多糖转运机制

• 批准号: 10328545
• 负责人: Keith Patrick Romano
• 金额: $ 19.98万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-03 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10328545
• 关键词: Advisory Committees; Affect; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Arabinose; Area; Award; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biology; Biosynthetic Proteins; Carbapenems; Carrier Proteins; Cell Membrane Permeability; Cell Wall; Cells; Chemical Structure; Chemicals; Chronic; Collaborations; Complement; Data; Data Set; Electron Microscopy; Electrostatics; Engineering; Environment; Enzymes; Exclusion; Foundations; Funding; Future; Gene Expression Profiling; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Goals; Gram-Negative Bacteria; Hand; Health; Human; Immune system; K-Series Research Career Programs; Lead; Lipopolysaccharide Biosynthesis Pathway; Lipopolysaccharides; Lung; Membrane; Mentors; Mentorship; Metabolic Pathway; Methods; Molecular Probes; Nosocomial Infections; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Physicians; Proteins; Pseudomonas aeruginosa; Pulmonology; Regulator Genes; Regulatory Pathway; Reporter; Research Personnel; Research Proposals; Resistance; Science; Scientist; Structure; Structure-Activity Relationship; Techniques; Testing; Therapeutic Agents; Training; Training Programs; Training Support; Translating; Work; bacterial genetics; base; career; cell growth; cellular targeting; cryogenics; drug candidate; drug development; drug discovery; efflux pump; genome-wide; human pathogen; improved; inhibitor; insight; knowledge base; lead candidate; lead optimization; mutant; new therapeutic target; novel; novel strategies; novel therapeutics; pathogenic bacteria; promoter; protein expression; response; screening; small molecule; statistics; structural biology; transcriptomics; transport inhibitor

PROJECT SUMMARY Pseudomonas aeruginosa poses a major threat to human health due to limited treatment options and its ability to become resistant to antibiotics. P. aeruginosa and other Gram-negative bacteria are particularly difficult to treat because their asymmetric outer membranes, comprising an electronegative matrix of lipopolysaccharide (LPS) in the outer leaflet, form an electrostatic barrier excluding most antibiotics. The candidate aims apply advanced genomic, genetic and chemical biological strategies to study this important human pathogen, both to develop novel therapeutic agents and to gain insights into the basic biology of vulnerable targets. In work in progress, recent target-focused, whole cell screening identified 128 small molecules hypothesized to kill P. aeruginosa by disrupting LPS transport to the outer membrane. In Aim 1, with small molecule hits in hand, the candidate proposes to develop these compounds by optimizing their activities and establishing their mechanisms of action. To this end, the candidate has already developed high- throughput gene expression profiling methods to identify and prioritize hits that induce transcriptional responses in LPS transport pathways. Preliminary data revealed one lead candidate, C0918, induced a transcriptional response remarkably similar to that of a known LPS transport inhibitor, demonstrating that mechanisms of action can be inferred by gene responses compared to those of known antibiotics. Drawing on his background in protein science, when putative target proteins emerge, the candidate outlines strategies for protein expression, purification, direct-binding studies, and structure determination by cryogenic electron microscopy. Lead compounds in Aim 1 will also serve as valuable molecular probes to investigate the regulatory pathways underpinning LPS biosynthesis and transport in Aim 2. The candidate will perform a genetic screen to discover LPS regulatory genes in P. aeruginosa by mutagenizing an engineered reporter strain, which encodes fluorescent proteins marking expression levels of key LPS synthesis and transport genes. To complemental screening efforts, the candidate will also characterize single and double mutants encoding regulated copies of these key genes in LPS biosynthesis and transport, aimed at determining phenotypic consequences when LPS biosynthetic intermediates buildup under conditions of high LPS synthesis but low transport. With the guidance of his mentor, Dr. Deb Hung, the candidate has developed a five-year training program to provide both the technical and didactic training necessary to become an independent physician-scientist focused on using small molecules to target LPS transport, while also gaining insights into its underlying regulatory machinery in P. aeruginosa. Importantly, this project will be overseen by a scientific advisory committee providing expertise in key areas of this proposal, including LPS biology, bacterial genetics, genomics, and chemical biology. Throughout the career development award period, the candidate will expand his knowledge base with complete didactic and hands-on training. The candidate will complete coursework in bioinformatics and statistics to help with analyzing genomic-wide datasets. This proposal therefore provides the necessary training and scientific foundation to achieve Dr. Romano's ultimate goal of becoming a RO1-funded physician-scientist who applies advanced genomic and chemical biological techniques to study and treat bacterial pathogens.

项目总结