RNA polymerase and oxidative stress mediate ceftriaxone resistance in Neisseria gonorrhoeae

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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.