Comprehensive genetic characterization of antibiotic resistance

描述（由申请人提供）：我们建议开发和应用一套全面的实验和计算方法，以揭示大肠杆菌抗生素耐受性的遗传基础。我们方法的核心是一种基于微阵列的遗传足迹技术，该技术提供了一个全球性的定量评估，评估基因组中的每个基因如何在抗生素暴露下对生存做出贡献。通过应用新的全基因组上位性分析框架，将确定的基因置于遗传和调控网络的背景下。我们的目标是探索轻度耐药性亚致死抗生素暴露和严重的耐受性，在“持久性”的背景下表示。初步研究为我们提出的框架提供了强有力的原则性证据。将该方法应用于E.大肠杆菌趋化性在数周的时间尺度上鉴定了95%的已知位点，揭示了这些位点到功能子模块中的组织，并鉴定了调节运动性的上下文依赖性表达的信号通路。此外，在一个已经被广泛探索了三十多年的表型中，我们发现了三十多个额外的新基因座，这些基因座通过不同的机制做出贡献，包括Rcs信号通路和环状二GMP第二信使系统。我们的方法应用于轻度和致命的抗生素暴露已经揭示了十几个基因座，其遗传扰动显着增加抗生素耐受性。拟议的工作有望显着扩大涉及的基因数量，并通过上位性，共表达和共遗传分析的辅助使用，使我们能够将这些基因的遗传和调控网络的背景下。我们希望我们的研究结果能从根本上推进对抗生素耐药性的理解，并为生物医学界提供表征良好的途径，作为新药开发的基础。 公共卫生相关性：抗生素耐药性正在迅速成为世界各地的主要健康危机。我们提出了一个全面的框架来研究不同药物类别耐药性的遗传基础。我们希望拟议的研究能够发现对抗抗生素耐药性的新途径。

DESCRIPTION (provided by applicant): We propose to develop and apply a comprehensive set of experimental and computational methods for revealing the genetic basis of antibiotic tolerance in Escherichia coli. At the core of our approach is a microarray-based genetic footprinting technology that provides a global quantitative assessment of how each and every gene in the genome contributes to survival under antibiotic exposure. The identified genes will be placed within the context of genetic and regulatory networks through the application of a novel genome-wide epistasis analysis framework. We aim to explore both mild resistance to sub-lethal antibiotic exposure and severe tolerance as expressed in the context of 'persistence'. Preliminary studies provide strong proof-of-principle evidence for the framework we propose. Application of our approach to E. coli chemotaxis identifies 95% of known loci on the time-scale of weeks, reveals the organization of these loci into functional sub-modules, and identifies signaling pathways that regulate the context-dependent expression of motility. Furthermore, in a phenotype that has been extensively explored for over thirty year, we find three dozen additional novel loci that contribute through diverse mechanisms including the Rcs signaling pathway and cyclic-di-GMP second messenger system. The application of our approach to mild and lethal antibiotic exposure has already revealed more than a dozen loci whose genetic perturbations dramatically increase antibiotic tolerance. The proposed work promises to significantly expand the number of genes involved, and through the adjunct use of epistasis, co-expression, and co-inheritance analysis, allow us to place these genes within the context of genetic and regulatory networks. We expect our findings to fundamentally advance the understanding of antibiotic resistance and to provide the biomedical community with well-characterized pathways that serve as the basis for the development of new drugs. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is rapidly becoming a major health crisis around the world. We propose a comprehensive framework for studying the genetic basis of resistance across diverse drug classes. We expect the proposed research to lead to the discovery of novel pathways for combating antibiotic resistance.