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 铜绿假单胞菌由于治疗选择有限及其 能够抗抗生素的能力。铜绿假单胞菌和其他革兰氏阴性细菌尤其是 由于其不对称的外膜，很难治疗，包括 外部小叶中的脂多糖（LPS），形成不包括大多数抗生素的静电屏障。这 候选人的目的应用高级基因组，遗传和化学生物学策略来研究这一重要 人类病原体，既可以发展新型的治疗剂，又有见解 脆弱的目标。在进行的工作中，最近以目标为中心的全细胞筛选确定了128个小 通过破坏LPS转运到外膜的运输，假设可以杀死铜绿假单胞菌的分子。在AIM 1中， 候选人手里拿着小分子的命中，提议通过优化它们来开发这些化合物 活动并建立他们的行动机制。为此，候选人已经发展了很高的 吞吐基因表达分析方法，以识别和优先级诱导转录的命中 LPS运输途径的响应。初步数据显示，一名主要候选人C0918诱导了A 转录响应与已知的LPS运输抑制剂非常相似，表明 与已知抗生素相比，基因反应可以推断作用机理。吸引 他在蛋白质科学方面的背景，当假定的靶蛋白出现时，候选人概述了策略 蛋白质表达，纯化，直接结合研究和低温电子的结构测定 显微镜。 AIM 1中的铅化合物还将作为研究调节途径的有价值的分子探针 AIM 2中的LPS生物合成和运输的基础。候选人将执行遗传筛查 通过对铜绿假单胞菌中的LPS调节基因进行诱变，以编码工程的报告菌株 荧光蛋白标记关键LPS合成和转运基因的表达水平。互补 筛选工作，候选人还将表征编码调节副本的单个和双重突变体 LPS生物合成和转运中的这些关键基因，旨在确定LPS时表型后果 生物合成中间体在高LPS合成但运输较低的条件下堆积。 Deb Hung博士在导师的指导下，候选人制定了一项为期五年的培训计划 提供成为独立医师科学家所需的技术和教学培训 专注于使用小分子以靶向LP的传输，同时也可以洞悉其基础 铜绿假单胞菌的监管机械。重要的是，该项目将受到科学咨询的监督 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。在整个职业发展奖中，候选人将扩大 他的知识基础通过完整的教学和动手培训。候选人将完成课程 生物信息学和统计数据，以帮助分析全基因组范围的数据集。因此，该建议 提供必要的培训和科学基础，以实现罗曼诺博士的最终目标 成为RO1资助的医师科学家，他应用高级基因组和化学 研究和治疗细菌病原体的生物学技术。