Targeting lipopolysaccharide transport machinery in Pseudomonas aeruginosa

项目总结 由于治疗选择有限，铜绿假单胞菌对人类健康构成了重大威胁 对抗生素产生抗药性的能力。铜绿假单胞菌和其他革兰氏阴性菌尤其 很难治疗，因为它们的不对称外膜，由一个电负性矩阵组成 外叶中的脂多糖(LPS)形成了一种静电屏障，排除了大多数抗生素。这个 候选人的目标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，既是为了开发新的治疗药物，也是为了深入了解 易受攻击的目标。在正在进行的工作中，最近针对目标的全细胞筛查发现了128个小细胞 假设分子通过破坏内毒素向外膜的运输来杀死铜绿假单胞菌。在目标1中， 随着小分子药物的问世，候选人建议通过优化它们的结构来开发这些化合物 活动，并建立其行动机制。为此，候选人已经发展得很高- 吞吐量基因表达谱方法用于识别和优先排序诱导转录的命中 内毒素转运途径中的反应。初步数据显示，一名领先的候选人C0918导致了 转录反应与已知的内毒素转运抑制剂非常相似，表明 与已知抗生素相比，可以通过基因反应来推断其作用机制。在上面绘制 他在蛋白质科学方面的背景，当假定的目标蛋白质出现时，候选人概述了 蛋白质的表达、纯化、直接结合研究和低温电子结构测定 显微镜。 目标1中的先导化合物也将作为有价值的分子探针来研究调控途径。 在AIM 2中支持内毒素的生物合成和运输。候选人将进行基因筛查以 通过突变一株编码内毒素的工程报告菌株来发现铜绿假单胞菌中的内毒素调节基因 荧光蛋白标记关键的内毒素合成和运输基因的表达水平。互为补充 在筛选工作中，候选人还将描述编码受调控拷贝的单突变体和双突变体的特征 这些在内毒素生物合成和运输中的关键基因，旨在确定当内毒素 生物合成中间体在高内毒素合成但低转运的条件下积累。 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，以 为成为一名独立的内科科学家提供必要的技术和教学培训 专注于使用小分子靶向脂多糖的运输，同时也获得了对其潜在的洞察力 铜绿假单胞菌的调控机制。重要的是，这个项目将由一个科学咨询机构监督 委员会提供这项提案的关键领域的专业知识，包括内毒素生物学、细菌遗传学、 基因组学和化学生物学。在整个职业发展奖励期间，候选人将扩大 他的知识基础和完整的说教和动手培训。这位候选人将在 生物信息学和统计学，帮助分析全基因组数据集。因此，这项提议 为实现罗曼诺博士的最终目标提供必要的培训和科学基础 成为一名应用先进基因组和化学技术的RO1资助的内科科学家 研究和治疗细菌病原体的生物技术。

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.