1. Microbiology and Infectious Disease
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RNA polymerase mutations cause cephalosporin resistance in clinical Neisseria gonorrhoeae isolates

  1. Samantha G Palace
  2. Yi Wang
  3. Daniel HF Rubin
  4. Michael A Welsh
  5. Tatum D Mortimer
  6. Kevin Cole
  7. David W Eyre
  8. Suzanne Walker
  9. Yonatan H Grad  Is a corresponding author
  1. Harvard T. H. Chan School of Public Health, United States
  2. Harvard Medical School, United States
  3. Public Health England, Royal Sussex County Hospital, United Kingdom
  4. Big Data Institute, University of Oxford, United Kingdom
  5. Brigham and Women’s Hospital, Harvard Medical School, United States
Research Article
Cite this article as: eLife 2020;9:e51407 doi: 10.7554/eLife.51407
6 figures, 3 tables, 4 data sets and 5 additional files

Figures

RNA polymerase-mediated reduced cephalosporin susceptibility among gonococcal isolates.

Maximum likelihood phylogeny of the 1102 isolates of the GISP dataset and one United Kingdom isolate (GCPH44). Most high-level reduced cephalosporin susceptibility (MIC ≥0.125 μg/mL) in this dataset is associated with the mosaic penA XXXIV allele, but some isolates with high MICs lack this allele. In four of these isolates – GCGS1095, GCGS1014, GCGS1013 (inset, red-marked leaves) and the U.K. isolate GCPH44 (orange arrow) – CRORS is caused by mutations in the RNA polymerase holoenzyme. Transformation of the CRORS allele rpoB1 from GCGS1095 confers phenotypic reduced susceptibility to the phylogenetic neighbor GCGS0457 (inset, pink-marked leaf) and to other susceptible clinical isolates (black arrowheads). The isolate GCGS0364 (gray arrowhead, denoted with *) spontaneously develops CRORS via rpoB mutation in vitro.

Location of CRORS-associated RNA polymerase mutations.

(A) Alignment of mutant RNA polymerase alleles associated with reduced ceftriaxone susceptibility. (B) Crystal structure of the RNA polymerase holoenzyme from E. coli by Zuo et al. (PDB 4YLO) (Zuo and Steitz, 2015), showing the location of the residues homologous to N. gonorrhoeae RpoB R201 (magenta), G158 (yellow), and P157 (orange). The flexible region of σ70 1.1 that includes the variant positions of rpoD1 and rpoD1 is not included in this structure, but is predicted to interact with this region of RpoB (Murakami, 2013).

Figure 3 with 1 supplement
RNA polymerase mutations do not change growth phenotypes.

(A) Growth of GCGS0457 and RNAP mutant transformants on solid GCB agar (mean and standard deviation of three technical replicates shown, representative of two independent experiments). RNAP mutations do not result in a growth rate defect. (B) Transmission electron micrographs of GCGS0457 and RNAP mutant transformants. CRORS transformants are slightly smaller than the parental GCGS0457 strain, but are otherwise morphologically similar.

Figure 3—figure supplement 1
Size and eccentricity of GCGS0457 and CRORS transformants.

100–105 cellular cross sections from TEM images were manually measured in ImageJ to calculate the (A) cross-sectional area and (B) eccentricity of cells from the CROS strain GCGS0457 and its CRORS transformants GCGS0457 RpoDE98K and GCGS0457 RpoBR201H. The cross-sectional area of CRORS transformant cells is slightly smaller (mean area: 0.585 μm2 for GCGS0457 RpoDE98K; 0.621 μm2 for GCGS0457 RpoBR201H) than cells of the parental strain GCGS0457 (mean area 0.693 μm2; p=3.977×10−5 compared to GCGS0457 RpoDE98K and p=0.00436 compared to GCGS0457 RpoBR201H by Welch’s t-test). The degree of eccentricity in the CRO cells is not significantly different between these populations.

Figure 4 with 3 supplements
Effect of CRORS-associated RNA polymerase mutations on PBP abundance.

(A) Normalized abundance of transcripts encoding penicillin binding proteins (PBPs) in parental isolates (GCGS0457, GCGS1095, GCGS1014) and RNAP mutant transformants in the GCGS0457 background, measured by RNAseq. (B) Relative protein abundance of PBP1, PBP2, and PBP3, as measured by bocillin-FL labeling. Total bocillin-FL signal for each strain was set at 100%. The relative contribution of each PBP to that signal is shown here. PBP4 protein was not observed on these gels, in agreement with previous reports (Stefanova et al., 2003).

Figure 4—figure supplement 1
Principle components analysis of RNA-seq data.

The first two principle components are shown for transcriptomic data collected from the CROS isolate GCGS0457, the CRORS isolates GCGS1095 and GCGS1014, and the CRORS transformants GCGS0457 RpoDE98K and GCGS0457 RpoBR201H (three biological replicates/strain). Replicates from both CRORS transformants cluster tightly, despite having different RNAP mutations.

Figure 4—figure supplement 2
Transcript abundance of various genes in CRORS strains with RNA polymerase mutations.

Normalized abundance of various transcripts in parental isolates (GCGS0457, GCGS1095, GCGS1014) and RNAP mutant transformants in the GCGS0457 background. Shown: mtrC, mtrD, and mtrE, which encode the components of the Mtr efflux pump; mtrR, which encodes the transcriptional repressor of the mtrCDE operon; ynhG, a putative L,D-transpeptidase; and pilQ, which encodes the pilus pore subunit PilQ.

Figure 4—figure supplement 3
Transcript abundance of various genes in CRORS strains with RNAP mutations, exposed to sub-inhibitory ceftriaxone.

Normalized abundance of various transcripts in parental isolates (GCGS0457, GCGS1095, GCGS1014) and RNAP mutant transformants, exposed to 0.008 μg/mL CRO for 90 min. Shown: mtrC, mtrD, and mtrE, which encode the components of the Mtr efflux pump; mtrR, which encodes the transcriptional repressor of the mtrCDE operon; ynhG, a putative L,D-transpeptidase; pilQ, which encodes the pilus pore subunit PilQ; and transcripts for each of the penicillin-binding proteins.

Effect of CRORS-associated RNA polymerase mutations on cell wall structure.

Relative abundance of muropeptide peaks in cell wall digests of GCGS0457 and its CRORS derivatives, GCGS0457 RpoDE98K and GCGS0457 RpoBR201H. Transformant cell walls contain a higher proportion of peptidoglycan with pentapeptide stems (peaks 4, 8, 15b, and 16). Relative abundance values for each muropeptide species (right) were calculated by extracting the peak mass from the total ion chromatogram, integrating the resulting peak area, and dividing by the sum of all of the peak areas within each sample. Shown: mean and standard deviation from 3 biological replicates (data from individual replicates can be found in Figure 5—source data 1). Muropeptide species that are significantly altered in abundance each transformant compared to the parental strain GCGS0457 are indicated in bold (p<0.05 by two-tailed t test). See Supplementary file 3 for a list of all muropeptide masses detected.

Figure 5—source data 1

Relative abundance of muropeptide species in individual biological replicates.

https://cdn.elifesciences.org/articles/51407/elife-51407-fig5-data1-v2.xlsx
Author response image 1

Tables

Table 1
Selected strains, phenotypic ceftriaxone susceptibility, and relevant genotypes.
strainCRO MIC (μg/mL)penA (PBP2)ponA (PBP1)PorB 120/121RpoBRpoD
GCGS04570.012Type IX non-mosaic (NG STAR 9.001)421P120G/121Vwildtypewildtype
GCGS10950.19Type IX non-mosaic (NG STAR 9.001)421P120N/121AR201H (rpoB1)wildtype
GCGS10140.125Type IX non-mosaic (NG STAR 9.001)421P120D/121AwildtypeE98K (rpoD1)
GCGS10130.19Type IX non-mosaic (NG STAR 9.001)421P120G/121AwildtypeΔ92–95 (rpoD2)
GCPH440.125Type II non-mosaic (NG STAR 2.001)421L120G/121AH553NΔ92–95 (rpoD2)
GCGS0457 rpoB10.19Type IX non-mosaic (NG STAR 9.001)421P120G/121VR201H (rpoB1)wildtype
GCGS0457 rpoD10.125Type IX non-mosaic (NG STAR 9.001)421P120G/121VwildtypeE98K (rpoD1)
GCGS0457 rpoD20.19Type IX non-mosaic (NG STAR 9.001)421P120G/121VwildtypeΔ92–95 (rpoD2)
GCGS03640.023Type IX non-mosaic (NG STAR 9.001)421P120K/121Dwildtypewildtype
GCGS0364 rpoB20.5Type IX non-mosaic (NG STAR 9.001)421P120K/121DG158V (rpoB2)wildtype
GCGS0364 rpoB30.75Type IX non-mosaic (NG STAR 9.001)421P120K/121DP157L (rpoB3)wildtype
Table 2
Antibiotic susceptibility profiles of CRORS strains with RNAP mutations.
StrainMIC (μg/ml)
CROCFXPENEPMAZITETCIPRIF
GCGS04570.0120.01620.0160.251≤0.0150.125
GCGS10950.19120.0320.51≤0.015≤0.0625
GCGS10140.1250.51.50.0320.251≤0.015≤0.0625
GCGS10130.190.51.50.0230.51≤0.015≤0.0625
GCGS0457 RpoBR201H0.19>0.520.0470.251≤0.015≤0.0625
GCGS0457 RpoDE98K0.1250.51.50.0230.251≤0.015≤0.0625
GCGS0457 RpoDΔ92-950.190.520.0230.251≤0.015≤0.0625
Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Neisseria gonorrhoeae)penANeisseria gonorrhoeae PubMLSTNEIS1753
Gene (Neisseria gonorrhoeae)ponANeisseria gonorrhoeae PubMLSTNEIS0414
Gene (Neisseria gonorrhoeae)rpoBNeisseria gonorrhoeae PubMLSTNEIS0123
Gene (Neisseria gonorrhoeae)rpoDNeisseria gonorrhoeae PubMLSTNEIS1466
Strain, strain background (Neisseria gonorrhoeae)GCGS0457(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS1095(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS1014(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS1013(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0364(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0092(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0126(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0161(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0249(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0275(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0336(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0353(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0374(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0465(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0489(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0497(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0501(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0524(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0560(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0576(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0698(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCGS0769(Grad et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)GCPH44(De Silva et al., 2016)
Strain, strain background (Neisseria gonorrhoeae)28BLGift of S. Johnson
Chemical compound, drugCeftriaxone disodium salt hemi(heptahydrate)Sigma AldrichCat. # C5793
Chemical compound, drugCeftriaxone EtestbioMérieuxSKU # 412302 and 412300
Chemical compound, drugPenicillin G EtestbioMérieuxSKU # 412262
Chemical compound, drugErtapenem EtestbioMérieuxSKU # 531640
Chemical compound, drugCefixime trihydrateSigma AldrichCat. # 18588
Chemical compound, drugAzithromycinSigma AldrichCat. # PHR1088
Chemical compound, drugTetracycline hydrochlorideSigma AldrichCat. # T7660
Chemical compound, drugCiprofloxacinSigma AldrichCat. # 17850
Chemical compound, drugRifampicinChem-ImpexCat. # 00260
Chemical compound, drugBocillin FL penicillin, sodium saltThermo FisherCat. # B13233
Sequence-based reagentRpoB-USThis paperPCR primersATGCCGTCTGAATATCAGATTGATGCGTACCGTT
Sequenced-based reagentRpoB-DSThis paperPCR primersCGTACTCGACGGTTGCCCAAG
Sequenced-based reagentRpoD-USThis paperPCR primersAACTGCTCGGACAGGAAGCG
Sequenced-based reagentRpoD-DSThis paperPCR primersCGCGTTCGAGTTTGCGGATGTT

Data availability

Sequencing data have been deposited in the NCBI SRA database under accession number PRJNA540288.

The following data sets were generated
  1. 1
    NCBI BioProject
    1. SG Palace
    2. Y Wang
    3. YH Grad
    (2019)
    ID PRJNA540288. N. gonorrhoeae isolates with reduced ceftriaxone susceptibility.
The following previously published data sets were used
  1. 1
    ENA
    1. YH Grad
    2. RD Kirkcaldy
    3. D Trees
    4. J Dordel
    5. SR Harris
    6. E Goldstein
    7. H Weinstock
    8. J Parkhill
    9. WP Hanage
    10. S Bentley
    11. M Lipsitch
    (2013)
    ID PRJEB2999. Emergence and spread of antibiotic resistant Neisseria gonorrhoeae in the US.
  2. 2
    ENA
    1. YH Grad
    2. SR Harris
    3. RD Kirkcaldy
    4. AG Green
    5. DS Marks
    6. SD Bentley
    7. D Trees
    8. M Lipsitch
    (2016)
    ID PRJEB7904 and PRJEB2090. Extended_genomic_epidemiology_of_Neisseria_gonorrhoeae_with_reduced_susceptibility_to_extended_spectrum_cephalosporins_and_azithromycin_in_the_US.
  3. 3
    NCBI Sequence Read Archive
    1. Silva D De
    2. J Peters
    3. K Cole
    4. MJ Cole
    5. F Cresswell
    6. G Dean
    7. J Dave
    8. D Rh Thomas
    9. K Foster
    10. A Waldram
    11. DJ Wilson
    12. X Didelot
    13. YH Grad
    14. DW Crook
    15. TEA Peto
    16. AS Walker
    17. J Paul
    (2016)
    ID SRR3360905. Whole-genome sequencing to determine Neisseria gonorrhoeae transmission: an observational study.

Additional files

Supplementary file 1

Allelic diversity of RNA polymerase holoenzyme components and sigma factors.

https://cdn.elifesciences.org/articles/51407/elife-51407-supp1-v2.xlsx
Supplementary file 2

Ceftriaxone susceptibility and genotype of isolates that have or can acquire CRORS-associated RNA polymerase mutation.

https://cdn.elifesciences.org/articles/51407/elife-51407-supp2-v2.docx
Supplementary file 3

Muropeptide masses detected in cell wall digests of GCGS0457 and RNAP mutants.

https://cdn.elifesciences.org/articles/51407/elife-51407-supp3-v2.docx
Supplementary file 4

N. gonorrhoeae strains used in this study.

https://cdn.elifesciences.org/articles/51407/elife-51407-supp4-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/51407/elife-51407-transrepform-v2.docx

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