Identification and analysis of compensatory mutations that support the evolution of antibiotic resistance in Neisseria gonorrhoeae

项目摘要 淋病奈瑟菌感染率的增加和抗生素耐药性的传播迫切需要 对公众健康的威胁。支持抗生素出现和传播的途径和基因的知识， 耳耐药性是发展新的监测、诊断和治疗策略所必需的。尽管我们 尽管人们对赋予抗性的基因和等位基因的了解有限，但关于抗性的基因和等位基因的知识仍存在重大差距。 导致淋球菌种属间耐药性分布不均匀的因素。核心 一个尚未充分探讨的问题是耐药决定因素对淋球菌适应性的影响：在多大程度上 抗性决定因素是否影响适应性，如果它们产生适应性成本，淋球菌如何适应， 降低这些成本？在这一建议中，我们通过一项将经验和能力结合起来的全面战略来解决这些差距， 补偿突变的心理和计算识别及其作用机制的研究。 该项目的总体目标是确定增加对这两种药物耐药性的突变的影响。 大多数临床相关的抗生素治疗淋病，环丙沙星和头孢曲松，对细菌的健身。 我们将通过三个具体目标实现这一目标。在目标1中，我们将确定抵抗的适应性成本 等位基因转化到不同生态位的感病菌株中时， 通过雌性小鼠的实验进化来减轻这些适应性成本的补偿性突变 模型我们将检测两种最常见的环丙沙星耐药等位基因（gyrAS 91 F，D95 G和 parCS 87 R）和4个头孢曲松耐药等位基因（佩纳的两种变体，致死性p53基因， 头孢曲松的靶点，以及新描述的rpoB和rpoD变体），并评估compen的依赖性。 在基因组背景上的饱和途径。在目标2中，我们将利用我们收集的7500多种淋球菌， 基因组从临床分离，我们有抗药性表型，并采用人口ge- 组学方法来识别潜在的补偿突变，并在小鼠模型中测试这些突变。而且我们 将定义目标1中确定的候选者的等位基因多样性和分布。在目标3中，我们将确定 目的1和2研究中确认的补偿性突变的作用机制， 研究生长和形态学、转录组学、代谢组学和定向研究的综合策略 生物化学功能。我们还将建立在acnB和mleN补偿突变的初步数据基础上， 头孢曲松耐药性和gyrA介导的喹诺酮耐药性中的硫胺素生物合成途径。 这个跨学科项目汇集了三个领导的互补和非重叠的专业知识， 在N.淋病，连接小鼠模型， 淋球菌感染（Jerse博士），人口基因组学（格拉德博士），以及生化和生理特征， 耐药相关变异的化（Nicholas博士）。

PROJECT SUMMARY The increasing rate of infection and spread of antibiotic resistance in Neisseria gonorrhoeae poses an urgent threat to public health. Knowledge of the pathways and genes that support the emergence and spread of antibi- otic resistance is necessary to develop new strategies for surveillance, diagnosis, and treatment. Despite our understanding of the genes and alleles that confer resistance, significant knowledge gaps remain regarding the factors that contribute to the uneven distribution of resistance across the gonococcal species phylogeny. A core issue that remains poorly explored is the impact of resistance determinants on gonococcal fitness: to what extent do resistance determinants impact fitness, and, if they incur a fitness cost, how does the gonococcus adapt and mitigate these costs? In this proposal, we address these gaps through a comprehensive strategy linking experi- mental and computational identification of compensatory mutations with studies of their mechanisms of action. The overall goal of this project is to determine the impact of mutations that increase resistance to the two most clinically relevant antibiotics for treatment of gonorrhea, ciprofloxacin and ceftriaxone, on bacterial fitness. We will achieve this goal through three specific aims. In Aim 1, we will determine the fitness costs of resistance alleles when transformed into susceptible isolates from different niches and with distinct phylogeny and identify compensatory mutations that mitigate these fitness costs through experimental evolution in the female mouse model. We will examine the two most common ciprofloxacin resistance-conferring alleles (gyrAS91F,D95G and parCS87R) in clinical isolates, and four ceftriaxone resistance-conferring alleles (two variants of penA, the lethal target of ceftriaxone, and newly described variants in rpoB and rpoD), and evaluate the dependence of compen- satory pathways on genomic background. In Aim 2, we will leverage our collection of over 7500 gonococcal genomes from clinical isolates for which we have antibiotic-resistance phenotypes and employ population ge- nomics methods to identify potential compensatory mutations and test these in the mouse model. Moreover, we will define the allelic diversity and distribution of candidates identified in Aim 1. In Aim 3, we will determine the mechanism of action of confirmed compensatory mutations arising from the studies in Aims 1 & 2 using an integrative strategy that examines growth and morphology, transcriptomics, metabolomics, and directed studies of biochemical function. We will also build on preliminary data on compensatory mutations in acnB and mleN for ceftriaxone resistance and on the thiamine biosynthesis pathway in gyrA-mediated quinolone resistance. This interdisciplinary project brings together the complementary and non-overlapping expertise of three lead- ing investigators in the biology and genetics of antibiotic resistance in N. gonorrhoeae, linking the mouse model of gonococcal infection (Dr. Jerse), population genomics (Dr. Grad), and biochemical and physiological charac- terization of resistance-related variants (Dr. Nicholas).