RNA polymerase and oxidative stress mediate ceftriaxone resistance in Neisseria gonorrhoeae

项目摘要/摘要 淋球菌是一种革兰氏阴性细菌，主要感染人类泌尿生殖道。尽管 一旦出现下降，美国淋病感染的发病率在过去几乎翻了一番 十年。同时，耐药菌株比例也在增加。 这一时期的淋病，包括对一线抗生素头孢曲松耐药的菌株。初级阶段 头孢曲松的靶标是青霉素结合蛋白2(PBP2)，这是一种催化青霉素与青霉素发生交联的酶 细胞壁中的肽聚糖。尽管大多数耐药性是由PBP2基因突变引起的，但另一种选择是 耐药机制是通过RNA聚合酶(RNAP)复合体的变化来调节的。这些突变发生在 RNAP的成分不影响活性，并足以产生抗性，尽管只在特定的 淋病奈瑟菌临床菌株的亚群。在初步工作中，我发现含有 这些RNA聚合酶复合体突变增加了与以下基因相关的转录产物 用RNA测序法(RNA-seq)测定抗氧化剂活性。在其他细菌物种中，抗生素通过 头孢菌素类药物与氧化应激的产生有关。 这项拟议工作的目标是调查头孢曲松耐药性与 淋球菌的氧化应激。RNA聚合酶突变可能通过 氧化应激反应。目标1遵循与头孢曲松耐药相关的抗氧化基因 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 描述了全基因组对头孢曲松耐药的贡献。 淋病。这将使用CRISPR干扰(CRISPRi)来识别 在头孢曲松存在下对生存有利或有害。最后，Aim 3检查了等位基因 在RNAP突变存在的情况下，临床菌株的多样性调节了生存能力和头孢曲松耐药性。 多个菌株的表型-基因型相关性将通过使用菌株- 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 对复杂的抗生素耐药表型的机理理解，定义方法工具 研究淋病奈瑟菌的遗传学，并可能为淋病的双重治疗寻找新的治疗靶点。 淋病。通过这项工作，我将发展实验和计算技能，作为我博士学位的一部分 论文在陈天海公共卫生学院Yonatan研究生的实验室进行研究。这项研究 计划将提高我独立构建和检验细菌基因组学和遗传学假说的能力 以减少抗生素耐药性对健康的影响。

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.