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).

项目总结 淋球菌不断增加的感染率和耐药性的传播构成了一个紧迫的问题 对公众健康的威胁。关于支持抗菌素出现和传播的途径和基因的知识- 耳源性耐药性对于制定新的监测、诊断和治疗策略是必要的。尽管我们 对于赋予抗性的基因和等位基因的了解，关于 导致淋球菌种系间耐药性分布不均的因素。一个核心 耐药决定因素对淋球菌适合性的影响：在多大程度上 耐药性决定因素是否影响健康，如果它们产生健康成本，淋球菌如何适应和 降低这些成本？在这份提案中，我们通过一项全面的战略来解决这些差距，该战略将经验与 对代偿突变的心理和计算识别及其作用机制的研究。 这个项目的总体目标是确定增加对这两种病毒耐药性的突变的影响。 临床上治疗淋病最相关的抗生素，环丙沙星和头孢曲松，对细菌适应性的影响。 我们将通过三个具体目标实现这一目标。在目标1中，我们将确定抵抗力的健康成本 将等位基因转化为来自不同生态位、具有不同系统发育和鉴定的敏感菌株 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 模特。我们将检测两个最常见的环丙沙星耐药等位基因(gyrAS91F、D95G和 临床分离株中的parCS87R)和四个头孢曲松耐药等位基因(Pena的两个变种，致命的 头孢曲松的靶点，以及新发现的rpoB和rpoD的变异体)，并评价其对药物的依赖性。 基因组背景下的转导途径。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耐药表型并使用群体gE-1。 识别潜在的补偿性突变并在小鼠模型中测试这些突变的常规方法。此外，我们 将定义目标1中确定的候选基因的等位基因多样性和分布。在目标3中，我们将确定 从AIMS 1和2中的研究中证实的补偿性突变的作用机制 研究生长和形态、转录组学、代谢组学和定向研究的综合战略 具有生物化学功能。我们还将建立关于acnB和mleN代偿性突变的初步数据 在gyrA介导的喹诺酮类耐药中对头孢曲松耐药和硫胺素生物合成途径的影响。 这一跨学科项目汇集了三家领先企业互补且互不重叠的专业知识- 淋球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)，以及生化和生理特性- 与抗药性相关的变异的术语(尼古拉斯博士)