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: 10544539
• 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/10544539
• 关键词: Advisory Committees; Affect; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Arabinose; Area; Award; Bacterial Antibiotic Resistance; Binding; Biochemical; Biochemistry; Bioinformatics; Biological; Biology; Biosynthetic Proteins; Carbapenems; Carrier Proteins; Cell Membrane Permeability; Cell Wall; Cells; Chemical Structure; Chemicals; Chronic lung disease; Collaborations; Complement; Cryoelectron Microscopy; Cytoplasm; Data; Data Set; 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; Medicine; Membrane; Mentors; Mentorship; Metabolic Pathway; Methods; Molecular Probes; Mutagens; 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; career; cell growth; cellular targeting; 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.

项目摘要 铜绿假单胞菌对人类健康构成重大威胁，这是由于有限的治疗选择及其 对抗生素产生耐药性。铜绿假单胞菌和其他革兰氏阴性菌特别是 难以治疗，因为它们的不对称外膜，包括 外叶中的脂多糖（LPS）形成排除大多数抗生素的静电屏障。的 候选人的目的是应用先进的基因组，遗传和化学生物学策略来研究这一重要的 人类病原体，既开发新的治疗药物，并获得深入了解的基础生物学 易受攻击的目标在正在进行的工作中，最近的靶向，全细胞筛选确定了128个小 分子假设通过破坏LPS转运到外膜来杀死铜绿假单胞菌。在目标1中， 随着小分子命中在手，候选人建议开发这些化合物，通过优化其 并建立其行动机制。为此，该候选人已经制定了高- 通过基因表达谱分析方法来鉴定和优先化诱导转录的命中， LPS运输途径的反应。初步数据显示，一个主要候选人，C 0918，诱导 转录反应与已知的LPS转运抑制剂的转录反应显著相似，表明 与已知抗生素相比，可以通过基因应答来推断作用机制。借鉴 他在蛋白质科学的背景，当假定的目标蛋白质出现，候选人概述了战略， 蛋白质表达、纯化、直接结合研究和通过低温电子显微镜测定结构 显微镜 Aim 1中的先导化合物也可作为研究调控途径的分子探针 支持Aim 2中的LPS生物合成和转运。候选人将进行基因筛查， 通过诱变工程报告菌株发现铜绿假单胞菌中的LPS调节基因， 标记关键LPS合成和转运基因表达水平的荧光蛋白。补充 筛选工作，候选人还将表征编码调控拷贝的单突变体和双突变体， 这些LPS生物合成和转运中的关键基因，旨在确定当LPS 在LPS合成高但转运低的条件下，生物合成中间体积累。 在他的导师Deb Hung博士的指导下，候选人制定了一个为期五年的培训计划， 提供必要的技术和教学培训，成为一个独立的物理学家，科学家 专注于使用小分子靶向LPS转运，同时也深入了解其潜在的 铜绿假单胞菌的调节机制。重要的是，这个项目将由一个科学顾问监督。 委员会在该提案的关键领域提供专业知识，包括LPS生物学，细菌遗传学， 基因组学和化学生物学。在整个职业发展奖期间，候选人将扩大 他的知识基础与完整的教学和动手训练。候选人将完成课程， 生物信息学和统计学，以帮助分析全基因组数据集。因此，这项建议 提供必要的培训和科学基础，以实现罗马诺博士的最终目标 成为一个RO 1资助的医生，科学家谁应用先进的基因组和化学 研究和治疗细菌病原体的生物技术。