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

支持淋病奈瑟菌抗生素耐药性进化的补偿突变的鉴定和分析

• 批准号: 10034093
• 负责人: Yonatan H Grad
• 金额: $ 79.62万
• 依托单位: HARVARD SCHOOL OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-17 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10034093
• 关键词: Active Sites; Address; Alleles; Anabolism; Animal Model; Animals; Antibiotic Resistance; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Back; Binding; Biochemical; Biology; Ceftriaxone; Cell physiology; Ciprofloxacin; Clinical; Coculture Techniques; Collection; Data; Dependence; Diagnosis; Diagnostic; Disease; Drug resistance; Essential Genes; Evolution; Female; Financial compensation; Genes; Genetic; Genetic Epistasis; Genome; Genomics; Goals; Gonorrhea; Growth; Guidelines; Human; In Vitro; Infection; Knowledge; Lethal Genes; Link; Maintenance; Mediating; Methods; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; Neisseria gonorrhoeae; Organism; Pathogenesis; Pathway interactions; Phenotype; Phylogeny; Physiological; Physiology; Population; Predisposition; Prevention; Public Health; Research Personnel; Resistance; Testing; Thiamine; Vaccines; Variant; bacterial fitness; base; clinically relevant; co-infection; cost; drug-resistant gonorrhea; experimental analysis; fitness; genome sequencing; improved; infection rate; insight; large datasets; metabolomics; mouse model; mutant; novel strategies; quinolone resistance; reproductive tract; resistance allele; resistance gene; resistance mutation; resistant strain; success; surveillance strategy; transcriptomics; whole genome

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).

项目概要 淋病奈瑟菌感染率的增加和抗生素耐药性的传播提出了一个紧迫的问题 对公众健康的威胁。了解支持抗生素出现和传播的途径和基因 耳阻力对于制定新的监测、诊断和治疗策略是必要的。尽管我们的 尽管对赋予抗性的基因和等位基因的了解，但在抗药性方面仍存在重大知识差距 导致淋球菌物种系统发育中耐药性分布不均匀的因素。一个核心 尚未充分探讨的问题是耐药性决定因素对淋球菌适应性的影响：影响到什么程度 抵抗力决定因素会影响健康吗？如果它们会产生健康成本，淋球菌如何适应和 减轻这些成本？在本提案中，我们通过一项综合战略来解决这些差距 补偿性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病毒的抵抗力的突变的影响 用于治疗淋病的最临床相关抗生素是环丙沙星和头孢曲松，对细菌适应性有影响。 我们将通过三个具体目标来实现这一目标。在目标 1 中，我们将确定阻力的适应成本 当转化为来自不同生态位且具有不同系统发育的易感分离株时，等位基因可被识别 通过雌性小鼠的实验进化来减轻这些适应成本的补偿性突变 模型。我们将检查两个最常见的环丙沙星耐药性等位基因（gyrAS91F、D95G 和 parCS87R），以及四个头孢曲松耐药性等位基因（penA 的两个变体，致命的 头孢曲松的目标，以及新描述的 rpoB 和 rpoD 变体），并评估补偿的依赖性 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 来自临床分离株的基因组，我们对其具有抗生素抗性表型并采用群体基因组 识别潜在补偿突变并在小鼠模型中测试这些突变的经济学方法。此外，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 的研究证实了补偿性突变的作用机制，使用 检查生长和形态、转录组学、代谢组学和定向研究的综合策略 的生化功能。我们还将建立关于 acnB 和 mleN 补偿突变的初步数据 头孢曲松耐药性以及gyrA介导的喹诺酮耐药性中硫胺素生物合成途径的影响。 这个跨学科项目汇集了三位领导者互补且不重叠的专业知识 淋病奈瑟菌抗生素耐药性的生物学和遗传学研究人员将小鼠模型联系起来 淋球菌感染（Jerse 博士）、群体基因组学（Grad 博士）以及生化和生理特征 耐药相关变异的特化（尼古拉斯博士）。