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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/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。尽管一度呈下降趋势，美国淋病感染发病率较过去几乎翻倍十年。耐抗生素菌株的比例同时增加。在此期间的淋病菌，包括对一线抗生素头孢曲松具有耐药性的菌株。初级头孢曲松的靶点是青霉素结合蛋白 2 (PBP2)，这是一种催化青霉素结合蛋白交联的酶。细胞壁中的肽聚糖。尽管大多数耐药性是由 PBP2 突变引起的，但另一种选择耐药机制是由 RNA 聚合酶 (RNAP) 复合物的变化介导的。这些突变在 RNAP 的成分不影响生存力并且足以赋予抗性，尽管仅在特定的情况下淋病奈瑟氏菌临床菌株的子集。在初步工作中我发现含有这些RNA聚合酶复合体突变导致与相关基因的转录物增加通过 RNA 测序 (RNA-seq) 检测抗氧化活性。在其他细菌物种中，抗生素介导的杀伤作用是头孢菌素与氧化应激的产生有关。拟议工作的目标是研究头孢曲松耐药性和淋病奈瑟菌的氧化应激。 RNA聚合酶突变可能通过以下方式导致耐药性：氧化应激反应。目标 1 追踪与头孢曲松耐药相关的抗氧化基因通过 RNA-seq 并研究这些靶基因在头孢曲松介导的细胞死亡中的作用。目标2 描述了 N. 突变体 RNAP 菌株中对头孢曲松耐药性的全基因组贡献。淋病。这将通过使用 CRISPR 干扰 (CRISPRi) 来识别基因来完成。头孢曲松存在时对生存有益或有害。最后，目标 3 检查等位基因如何在 RNAP 突变存在的情况下，临床菌株的多样性介导活力和头孢曲松耐药性。将通过使用菌株-基因型相关性同时研究多个菌株的表型-基因型相关性。特定条形码和微生物全基因组关联研究（GWAS）。这些发现将提供对复杂抗生素耐药表型的机制理解，定义方法工具对淋病奈瑟菌遗传学的研究，可能会确定淋病奈瑟菌双重治疗的新治疗靶点。淋病。通过这项工作，我将培养实验和计算技能，作为我博士课程的一部分在 T.H. Yonatan Grad 实验室进行论文研究陈公共卫生学院。这项研究计划将提高我独立构建和测试细菌基因组学和遗传学假设的能力减少抗生素耐药性对健康的影响。