RNA polymerase and oxidative stress mediate ceftriaxone resistance in Neisseria gonorrhoeae

RNA聚合酶和氧化应激介导淋病奈瑟菌头孢曲​​松耐药性

• 批准号: 10614613
• 负责人: Daniel Hidemitsu Fujii Rubin
• 金额: $ 5.27万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10614613
• 关键词: Affect; Alleles; Antibiotic Resistance; Antibiotics; Antioxidants; Bar Codes; CRISPR interference; Ceftriaxone; Cell Death; Cell Wall; Cephalosporin Resistance; Cephalosporins; Cervicitis; Clinical; Complex; DNA-Directed RNA Polymerase; Data Set; Databases; Dependence; Drug Combinations; Electron Transport; Enzymes; Ethnic Origin; Fright; Gene Mutation; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genomics; Genotype; Goals; Gonorrhea; Gram-Negative Bacteria; Growth; Health; Holoenzymes; Human; Incidence; Individual; Infection; Investigation; Knock-out; Laboratories; Libraries; Locales; Measures; Mediating; Methodology; Methods; Minimum Inhibitory Concentration measurement; Modeling; Monitor; Multi-Drug Resistance; Mutation; Neisseria gonorrhoeae; Oxidative Stress; Pathway interactions; Pelvic Inflammatory Disease; Penicillin Binding Protein 2; Peptidoglycan; Pharmaceutical Preparations; Phenotype; Point Mutation; Predisposition; Production; Proteins; Public Health Schools; Reactive Oxygen Species; Reporter; Research; Resistance; Role; Sexually Transmitted Diseases; Single Nucleotide Polymorphism; Superbug; Technology; Testing; Transcript; United States; Urethritis; Work; age group; biological adaptation to stress; crosslink; design; efflux pump; genome wide association study; genome-wide; iron metabolism; knock-down; loss of function; microbial genome; mosaic variant; mutant; new technology; new therapeutic target; next generation sequencing; novel; nuclease; overexpression; pathogen; resistance mechanism; resistant strain; response; skills; tool; transcriptome sequencing; urogenital tract

PROJECT SUMMARY/ABSTRACT Neisseria gonorrhoeae is a Gram negative bacterium that primarily infects the human urogenital tract. Though once on the decline, the incidence of gonorrheal infection in the United States has almost doubled over the past decade. There has been a simultaneous increase in the proportion of antibiotic resistant strains of N. gonorrhoeae over this period, including strains that are resistant to the first-line antibiotic ceftriaxone. The primary target of ceftriaxone is penicillin-binding protein 2 (PBP2), an enzyme that catalyzes the crosslinking of peptidoglycan in the cell wall. Though most resistance is caused by mutations in PBP2, one alternative mechanism of resistance is mediated by changes in the RNA polymerase (RNAP) complex. These mutations in components of RNAP do not affect viability and are sufficient to confer resistance, though only in a specific subset of clinical strains of Neisseria gonorrhoeae. In preliminary work I have found that strains that contain these RNA polymerase complex mutations have an increase in transcripts from genes associated with antioxidant activity by RNA sequencing (RNA-seq). In other bacterial species, antibiotic-mediated killing by cephalosporins has been associated with the production of oxidative stress. The goal of the proposed work is to investigate the relationship between ceftriaxone resistance and oxidative stress in N. gonorrhoeae. RNA polymerase mutations may contribute to resistance through the oxidative stress response. Aim 1 follows the antioxidant genes that are associated with ceftriaxone resistance by RNA-seq and investigates the role of these target genes in ceftriaxone-mediated cell death. Aim 2 characterizes the genome-wide contributions to ceftriaxone resistance in mutant RNAP strains of N. gonorrhoeae. This will be accomplished using CRISPR interference (CRISPRi) to identify genes that are beneficial for or deleterious to survival in the presence of ceftriaxone. Finally, Aim 3 examines how allelic diversity across clinical strains mediates viability and ceftriaxone resistance in the presence of RNAP mutations. Phenotype-genotype correlations in multiple strains will be investigated simultaneously through the use of strain- specific barcodes and microbial genome-wide association studies (GWAS). These findings will provide mechanistic understandings for a complex antibiotic resistance phenotype, define methodological tools for the investigation of N. gonorrhoeae genetics, and may identify novel therapeutic targets for dual treatment of N. gonorrhoeae. Through this work, I will develop experimental and computational skills as part of my doctoral dissertation studies in the laboratory of Yonatan Grad at the T.H. Chan School of Public Health. This research plan will advance my ability to independently construct and test hypotheses with bacterial genomics and genetics to reduce the health impact of antibiotic resistance.

项目摘要/摘要 淋病奈瑟氏菌是一种革兰氏阴性细菌，主要感染人类泌尿生殖道。尽管 一旦下降，美国淋病感染的发病率几乎翻了一番 十年。 N。 在此期间，淋病，包括对一线抗生素头孢曲松具有抗性的菌株。主要 头孢曲松的靶标是青霉素结合蛋白2（PBP2），这是一种催化交联的酶 肽聚糖在细胞壁中。尽管最大的阻力是由PBP2突变引起的，但一种替代 电阻机理是由RNA聚合酶（RNAP）复合物的变化介导的。这些突变中 RNAP的组成部分不会影响生存能力，并且足以赋予抵抗力，尽管仅在特定中 淋病奈瑟氏菌临床菌株的子集。在初步工作中，我发现包含的菌株 这些RNA聚合酶复合酶复合物突变的转录本与与之相关的基因的转录本增加 RNA测序（RNA-SEQ）的抗氧化活性。在其他细菌物种中，抗生素介导的杀伤 头孢菌素与氧化应激的产生有关。 拟议工作的目的是调查头孢曲松抗性与 淋病链球菌中的氧化应激。 RNA聚合酶突变可能有助于通过 氧化应激反应。 AIM 1遵循与头孢曲松抗性相关的抗氧化基因 通过RNA-Seq，研究了这些靶基因在头孢曲松介导的细胞死亡中的作用。目标2 表征了全基因组对N。 淋病。这将使用CRISPR干扰（CRISPRI）来识别是 有益于或有害在头孢曲松存在下生存。最后，AIM 3检查了等位基因 在RNAP突变存在下，临床菌株之间的多样性介导了生存能力和头孢曲松抗性。 多种菌株中的表型基因型相关性将通过菌株同时研究 特定的条形码和微生物基因组关联研究（GWAS）。这些发现将提供 对复杂抗生素抗性表型的机械理解，定义了方法学工具 研究淋病链球菌遗传学，并可能鉴定出新的治疗靶标，用于双重治疗N。 淋病。通过这项工作，我将发展实验和计算技能，作为我的博士学位的一部分 T.H. Yonatan毕业实验室的论文研究陈公共卫生学院。这项研究 计划将提高我使用细菌基因组学和遗传学独立构建和检验假设的能力 降低抗生素耐药性的健康影响。