Abstract
The evolution of the obligate human pathogen Neisseria gonorrhoeae has been shaped by selective pressures from diverse host niche environments and antibiotics. The varying prevalence of antibiotic resistance across N. gonorrhoeae lineages suggests that underlying metabolic differences may influence the likelihood of acquisition of specific resistance mutations. We hypothesized that the requirement for supplemental CO2, present in approximately half of isolates, reflects one such example of metabolic variation. Here, using a genome-wide association study and experimental investigations, we show that CO2 dependence is attributable to a single substitution in a β-carbonic anhydrase, CanB. CanB19E is necessary and sufficient for growth in the absence of CO2, and the hypomorphic CanB19G variant confers CO2 dependence. Furthermore, ciprofloxacin resistance is correlated with CanB19G in clinical isolates, and the presence of CanB19G increases the likelihood of acquisition of ciprofloxacin resistance. Together, our results suggest that metabolic variation has affected the acquisition of fluoroquinolone resistance.
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Data availability
The data sets generated during and/or analysed in this study can be found in the source data associated with this work. RNA-seq reads are available publicly at SRA Bioproject PRJNA869861. The NCCP11945 genome is accessible via RefSeq accession NC_011035.1. Genomic data, metadata and accession numbers are publicly available in previously published work69. Source data are provided with this paper.
Code availability
Code used in this study has been published69.
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Acknowledgments
We thank the members of the Grad Laboratory and the Waldor Laboratory for their invaluable feedback; the members of the gonococcal subgroup, particularly A. Bandekar, for their invaluable contributions; S. Palace for her thoughts on experimental design; and the Microbial Genome Sequencing Center (https://www.migscenter.com/) and SeqCenter (https://www.seqcenter.com/) for their work on sequencing strains. This work was supported by grants NIH R01 AI132606 and R01 AI153521 and by the Smith Family Foundation Odyssey award (Y.H.G.) and R01 AI 042347-24 (M.K.W.). Authors are further funded by grants NIH F30 AI160911-01 (D.H.F.R.), NIH T32 GM007753 (D.H.F.R.), NIH F31 AI156949-01 (K.H.) and NIH T32 AI132120-01.
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D.H.F.R. and K.C.M. performed the GWAS and statistical analyses. D.H.F.R. and K.A.W. performed the anaerobic experiments. D.H.F.R. and K.H. performed the macrophage experiments. D.H.F.R. performed the remainder of the experimental work. All authors (D.H.F.R., K.C.M., K.A.W., K.H., M.K.W. and Y.H.G.) contributed to data interpretation. Y.H.G. supervised and managed the study. D.H.F.R. and Y.H.G. wrote the manuscript. All authors reviewed and edited the final manuscript. All authors were responsible for the decision to submit for publication.
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Y.H.G. is on the scientific advisory board of Day Zero Diagnostics and consulted for GlaxoSmithKline. Y.H.G. has received funding from Merck and Pfizer. None of these competing interests has a bearing on this project. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Undirected genetic approaches identify a variant of CanB as causative for CO2-dependence.
(a) Schematic of undirected transformation of FA19 (parental CanB19G) to identify causative factors for CO2-dependence. (b) Plating efficiency in the presence and absence of CO2 following undirected transformation of gDNA from N. gonorrhoeae strain FA6140 into the N. gonorrhoeae lab strain FA19 (N = 10, 10, 9 from left to right from two independent experiments, error bars represent SEM). Significance (from left to right, p = 0.00018, p = 0.00028) determined by two-sided Mann-Whitney U test. (c) Putative reaction catalyzed by NGO2079/CanB and downstream metabolic products. (d) SNPs present in whole-genome sequencing of two transformants in (b). Arrow indicates the SNP identified in Fig. 1. (e) (Top) Alphafold predicted homodimeric structures of the CanB19E variant (teal) and the CanB19G variant (green). (Bottom) Magnified Alphafold predicted structures overlaying the glutamate (maroon) and glycine (red) at position 19. (f) Proportion of sequenced isolates by year with the CanB19G variant. *p < 0.05, **p < 0.01, ***p < 0.001. Figure created with BioRender.com.
Extended Data Fig. 2 The CanB19E variant has an advantage in the absence of CO2.
(a) Plating efficiency at different CO2 concentrations for N. gonorrhoeae strains 28BL (parental CanB19E) and FA19 (parental CanB19G) with isogenic CanB mutants (N = 6 for 0.1% CO2, N = 3 for air, from two independent experiments, error bars represent SEM). (b) Competition experiment in the presence of CO2 between FA19 CanB19G and FA19 CanBE19G with (left) the FA19 CanB19G strain kanamycin-labeled and (right) the FA19 CanBE19G kanamycin-labeled. The proportion of colony forming units (CFUs) that are kanamycin resistant is graphed against time (N = 3, representative of two independent experiments, error bars represent SEM). (c) Similar to (b), timecourse of competition between isogenic CanB FA19 strains in the presence of media supplementation and the absence of supplemental CO2 (N = 3, representative of two independent experiments, error bars represent SEM). (d) As in (c), competition between unlabeled FA19 CanBG19E and kanamycin-labeled FA19 CanB19G with selected metabolite supplementation assayed at 16 hours in the absence of supplemental CO2 (N = 6, from two independent experiments, error bars represent SEM). (e) Western blot of E. coli MG1655Δcan complemented with IPTG-inducible CanB variants C-terminally FLAG-tagged with G6PD as loading control (representative of two independent experiments).
Extended Data Fig. 3 The 19E variant of NGO2079 is not advantaged at moderately low pH or within macrophages.
The 19E variant of NGO2079 is not advantaged at moderately low pH or within macrophages. (a) Gentamicin intracellular protection assay in RAW 264.7 macrophages as measured by CFUs (N = 3/timepoint, representative of two independent experiments, error bars represent SEM). (b) Competition experiment as in Extended Data Fig. 2b in pH-adjusted Graver-Wade media (N = 3, error bars represent SEM).
Extended Data Fig. 4 CanB19E and CanB19G isogenic pairs have similar MICs.
(a) MICs for sulfamethoxazole, trimethoprim, and trimethoprim/sulfamethoxazole for the FA19 and 28BL pairs isogenic CanB variants. (b) Sulfamethoxazole susceptibility of MG1655Δcan complemented with isogenic CanB variants and induced with IPTG. (c) MICs for clinically relevant antibiotics for FA19 CanB19G and FA19 CanBE19G as determined by E-test and agar dilution plating. (d) gyrA mutations for spontaneous ciprofloxacin escapees or clean mutants of FA19 CanB19G and FA19 CanB19E, along with associated ciprofloxacin MIC.
Extended Data Fig. 5 Loss of CanB does not appear to confer a loss of natural competence.
Loss of CanB does not appear to confer a loss of natural competence. (a) Plating efficiency in the presence and absence of CO2 for a knockout of CanB in the N. gonorrhoeae lab strain FA1090 (N = 6, from two independent experiments, error bars represent SEM). Significance (p = 0.0006) by two-sided Mann-Whitney U. (b) Natural competence of strains of N. gonorrhoeae as measured by uptake of a nalidixic acid resistance-conferring integrating plasmid (N = 3, representative of two independent experiments, error bars represent SEM). (c and d) As in (b), natural competence as measured by (c) uptake of KanR-conferring plasmid DR1 and (d) a PCR product containing the allele encoding for GyrA91F/95G (N = 6, representative of two independent experiments, error bars represent SEM). *p < 0.05, **p < 0.01, ***p < 0.001.
Extended Data Fig. 6 The CanB19G variant is associated with ciprofloxacin resistance across countries and does not lead to a hypermutator phenotype.
(a) Violin plots of drug MICs for ~10,000 N. gonorrhoeae clinical isolates. Black bar represents median, dotted lines represent 25th/75th percentiles. (b) Average log2 of ciprofloxacin MIC in µg/mL by country and by allele of CanB. Weighted average of all datasets is represented by black dots/line. Size of data point represents size of dataset. Error bars represent standard deviation as determined by weighted variance. Significance (p = 0.0120) by unpaired two-sided t-test. (c) Calculated mutation rates of resistance acquisition for to rifampin by fluctuation analysis of isogenic CanB strains. (N = 192, representative of two independent experiments, error bars represent 95% confidence interval). Significance (p = 0.029) by unpaired two-sided t-test (N = 192, representative of two independent experiments, error bars represent SD). (d) Cumulative distribution of mutants in experiments in (c) along with data on the inoculum size and final population size. *p < 0.05, **p < 0.01, ***p < 0.001.
Extended Data Fig. 7 The CanB19G variant does not affect killing by ciprofloxacin, but CanB19G provides an advantage in the presence of gyrA mutations across multiple gyrA alleles and strain backgrounds.
The CanB19G variant does not affect killing by ciprofloxacin, but CanB19G provides an advantage in the presence of gyrA mutations across multiple gyrA alleles and strain backgrounds. (a) Kill curve of isogenic CanB FA19 at 2x MIC and 32x MIC (N = 3, representative of two independent experiments). (b) Doubling time of replicates in Fig. 3e as determined by linear regression on log-transformed CFU/mL counts from 2–10 hours of growth (N = 12, from two independent experiments, error bars represent SEM). Significance (p = 1.9e-5) by unpaired two-sided t-test. (c) Growth of CanB isogenic isogenic 28BL strains bearing ciprofloxacin resistance-determining GyrA91F/95G (N = 3, representative of two independent experiments). Significance determined by unpaired two-sided t-test. (d) Competition between spontaneously ciprofloxacin resistant isogenic CanB strains (see Extended Data Fig. 2) and susceptible parental strains (N = 3, representative of two independent experiments). (e) Growth curves of CanB isogenic FA19 strains with spontaneous ciprofloxacin resistance-determining gyrA alleles (N = 3, representative of two independent experiments). (f and g) Competition between (f) FA19 CanBG19E and FA19 CanBG19E GyrA91F (N = 6, except UMP and 100 µM Adenosine, N = 4) and (g) FA19 CanB19G and FA19 CanB19G GyrA91F (N = 3) after 16 hours along with media supplementation in liquid GCP-K (representative of two independent experiments, error bars represent SEM). Significance (from left to right, p = 0.0002, p = 5.7e-13, p = 3.86e-5), by one-way ANOVA with Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.
Extended Data Fig. 8 The relationship between CanB19E and CO2-dependence depends on the acyl-ACP synthetase AasN in the PenA34 lineage of N. gonorrhoeae.
(a) Plating efficiency in the absence and presence of supplemental CO2 of clinical N. gonorrhoeae isolate NY0195 following the introduction of mutations in CanB and AasN (N = 6, from two independent experiments, error bars represent SEM). Significance (from left to right, p = 0.00034 p = 0.00028) by two-sided Mann-Whitney U test. (b) Maximum-likelihood tree as in Fig. 1a overlaid with tracks indicating AasN allele and the ceftriaxone resistance determinant mosaic PenA34 allele. *p < 0.05, **p < 0.01, ***p < 0.001.
Supplementary information
Supplementary Table 1
RNA-seq DESeq2 results.
Supplementary Table 2
Strains, plasmids and primers in this study.
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Rubin, D.H.F., Ma, K.C., Westervelt, K.A. et al. CanB is a metabolic mediator of antibiotic resistance in Neisseria gonorrhoeae. Nat Microbiol 8, 28–39 (2023). https://doi.org/10.1038/s41564-022-01282-x
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DOI: https://doi.org/10.1038/s41564-022-01282-x
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