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来揭示大肠杆菌抗生素耐受性的遗传基础。我们方法的核心是基于微阵列的遗传足迹技术，该技术提供了基因组中每个基因如何在抗生素暴露下有助于生存的全球定量评估。通过应用新的全基因组上位分析框架，将鉴定出的基因置于遗传和调控网络的背景下。我们的目标是探索对亚致死抗生素暴露的轻度耐药和在“持久性”背景下表达的严重耐受性。初步研究为我们提出的框架提供了强有力的原理证明。我们的方法在大肠杆菌趋化性上的应用在时间尺度上确定了95%的已知基因座，揭示了这些基因座在功能子模块中的组织，并确定了调节环境依赖性运动性表达的信号通路。此外，在一个已经被广泛探索了30多年的表型中，我们发现了36个额外的新基因座，这些基因座通过不同的机制起作用，包括Rcs信号通路和cyclic-di-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多个基因座，其遗传扰动显着增加抗生素耐受性。这项工作有望显著扩大所涉及的基因数量，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置于遗传和调控网络的背景下。我们希望我们的发现能够从根本上促进对抗生素耐药性的理解，并为生物医学界提供充分表征的途径，作为开发新药的基础。

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.