Mechanistic Basis for Decreased Antimicrobial Susceptibility in a Clinical Isolate of Neisseria gonorrhoeae Possessing a Mosaic-Like mtr Efflux Pump Locus

Abstract

Recent reports suggest that mosaic-like sequences within the mtr (multiple transferable resistance) efflux pump locus of Neisseria gonorrhoeae, likely originating from commensal Neisseria sp. by transformation, can increase the ability of gonococci to resist structurally diverse antimicrobials. Thus, acquisition of numerous nucleotide changes within the mtrR gene encoding the transcriptional repressor (MtrR) of the mtrCDE efflux pump-encoding operon or overlapping promoter region for both along with those that cause amino acid changes in the MtrD transporter protein were recently reported to decrease gonococcal susceptibility to numerous antimicrobials, including azithromycin (Azi) (C. B. Wadsworth, B. J. Arnold, M. R. A. Satar, and Y. H. Grad, mBio 9:e01419-18, 2018, https://doi.org/10.1128/mBio.01419-18). We performed detailed genetic and molecular studies to define the mechanistic basis for why such strains can exhibit decreased susceptibility to MtrCDE antimicrobial substrates, including Azi. We report that a strong cis-acting transcriptional impact of a single nucleotide change within the -35 hexamer of the mtrCDE promoter as well gain-of-function amino acid changes at the C-terminal region of MtrD can mechanistically account for the decreased antimicrobial susceptibility of gonococci with a mosaic-like mtr locus.IMPORTANCE Historically, after introduction of an antibiotic for treatment of gonorrhea, strains of N. gonorrhoeae emerge that display clinical resistance due to spontaneous mutation or acquisition of resistance genes. Genetic exchange between members of the Neisseria genus occurring by transformation can cause significant changes in gonococci that impact the structure of an antibiotic target or expression of genes involved in resistance. The results presented here provide a framework for understanding how mosaic-like DNA sequences from commensal Neisseria that recombine within the gonococcal mtr efflux pump locus function to decrease bacterial susceptibility to antimicrobials, including antibiotics used in therapy of gonorrhea.

Keywords: antibiotic resistance; efflux; gonorrhea; molecular genetics; transformation.

FIG 1

Shown are the nucleotide and amino acid sequence percent identities of genes and intervening regions of the mtr locus possessed by gonococcal strains with respect to FA19 (CP012026.1). Accession numbers are provided in Table S1. Clustal Omega multiple sequence alignments were performed using N. gonorrhoeae strains FA19, CDC2, FA1090, H041, and MS11. Alignments were generated for each mtr gene using their nucleotide and amino acid sequences, and nucleotide sequences were aligned for the intergenic regions. Pairwise identity matrices were calculated, and the pairwise identity values for each alignment are shown.

FIG 2

Shown are levels of expression of mtrR and mtrE genes in gonococcal strains FA19 and CDC2 (A), FA19 and CR.100 (B), FA19 and CR.101 (C), and FA19 and CR.102 (D). Gene transcript levels were quantified by qRT-PCR performed in triplicate with three biological replicates. Results are presented as average NER (normalized expression ratio) values (±SD) with P values. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 3

(A) Shown is the region of the mtrR-mtrC that was PCR amplified from chromosomal DNA of strain CDC2 (blue line) used to transform strain FA19. The region of recombination in transformant strain CR.100 is shown by a blue line. The nucleotide sequences of the mtrR/mtrC promoter region (mtrCDE coding strand) from strains FA19, CDC2, CR.100, and CR.101 are shown below. The translation start codon for mtrR is shown in green. The −10 and −35 hexamers of the mtrR and mtrCDE promoters are boxed. The TSS sites for both promoters are shown by asterisks. The red arrow shows the point mutation in the −35 hexamer of the mtrCDE sigma-70 promoter. Differences in nucleotide sequence or deletions are highlighted in red. (B) The predicted amino acid sequences of MtrR produced by strains FA19, CDC2, CR.100, CR.101, and CR.102 are shown. Differences at sites 79, 183, and 197 are highlighted in red and with asterisks.

FIG 4

(A) Shown are levels of MtrR repressor protein in whole-cell lysates of gonococcal strains as determined by Western immunoblotting. (B) The SDS-PAGE gel stained with CBB showing near-equivalent levels of protein (15 μg) loaded in each well is shown. Gonococcal strains are identified at the top of each well.

FIG 5

Shown are specific activities of β-Gal produced by gonococcal strain CR.102 (MtrR D79N) from pLES94 constructs without P_mtrC-lacZ (CR.102pLES; control), or with P_mtrC-lacZ with the CDC2 promoter (CR.102pLES2.2) or the same but with the WT −35 hexamer possessed by FA19 (CR.102pLES4.1). The results are shown as average values (±SD) with P values from three biologic replicates with each performed in triplicate. **, P ≤ 0.01.

FIG 6

Shown are the sequences of the MtrD protein produced by gonococcal strains as deduced by DNA sequencing. Amino acid differences of MtrD from strains CDC2, CR.100, CR.103, and CR.104 compared to FA19 are shown in green, with amino acids predicted to be sites for binding antimicrobials shown by red asterisks.