RNA polymerase and oxidative stress mediate ceftriaxone resistance in Neisseria gonorrhoeae

项目总结/摘要 淋病奈瑟菌是一种革兰氏阴性细菌，主要感染人类泌尿生殖道。虽然 美国淋病感染的发病率一度下降，但在过去几乎翻了一番 十年同时，耐药菌株的比例也在增加。 淋病，包括对一线抗生素头孢曲松耐药的菌株。主 头孢曲松的靶点是青霉素结合蛋白2（PBP 2），这是一种催化 细胞壁中的肽聚糖。尽管大多数耐药性是由PBP 2突变引起的，但另一种选择是， 抗性机制是由RNA聚合酶（RNAP）复合物的变化介导的。中的这些突变 RNAP的组分不影响生存力，并且足以赋予抗性，尽管仅在特定的 淋球菌临床菌株的子集。在初步工作中，我发现含有 这些RNA聚合酶复合体突变增加了与以下相关基因的转录本： 抗氧化活性通过RNA测序（RNA-seq）。在其他细菌物种中， 头孢菌素与氧化应激的产生有关。 这项工作的目的是研究头孢曲松耐药与 氧化胁迫淋病RNA聚合酶突变可能通过以下途径导致耐药性： 氧化应激反应AIM 1追踪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。目的2 描述了N. RNAP突变株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）来识别与基因表达相关的基因来实现。 在头孢曲松存在下对存活有益或有害。最后，Aim 3研究了等位基因 在RNAP突变存在的情况下，临床菌株的多样性介导了生存力和头孢曲松耐药性。 将通过使用菌株- 特异性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械理解，为 N.淋病遗传学，并可能确定新的治疗靶点双重治疗的N。 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。 在T.H. Yonatan格拉德的实验室进行论文研究。公共卫生学院。本研究 该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。

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.