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对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基因足迹技术，它提供了对基因组中的每一个基因在抗生素暴露下如何促进生存的全球定量评估。通过应用一种新的全基因组上位性分析框架，已确定的基因将被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性抗生素暴露的轻微耐药性和在“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则证明证据。将我们的方法应用于大肠杆菌的趋化作用，在几周的时间尺度上识别了95%的已知基因座，揭示了这些基因座到功能子模块的组织，并识别了调节运动性的上下文相关表达的信号通路。此外，在一个已经被广泛研究了30多年的表型中，我们发现了另外30多个新的基因座，它们通过不同的机制发挥作用，包括RCS信号通路和环二GMP第二信使系统。我们的方法在轻微和致命的抗生素暴露中的应用已经揭示了十几个基因座，它们的基因扰动显著增加了抗生素的耐受性。这项拟议的工作承诺显著扩大涉及的基因数量，并通过上位性、共表达和共同遗传分析的辅助使用，允许我们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将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具有良好特征的途径，作为新药开发的基础。

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.