Clinically relevant mutations that cause derepression of the Neisseria gonorrhoeae MtrC-MtrD-MtrE Efflux pump system confer different levels of antimicrobial resistance and in vivo fitness

Abstract

The MtrC-MtrD-MtrE efflux pump system confers resistance to macrolide antibiotics and antimicrobial substances of the host innate defence. Clinical isolates with increased resistance to erythromycin and azithromycin frequently harbour mutations in the mtrR structural gene, which encodes a repressor of the mtrCDE operon, or the mtrR promoter region. The MtrC-MtrD-MtrE system is important for gonococcal survival in the murine genital tract, and derepression of the mtrCDE operon via deletion of mtrR confers increased fitness in vivo. Here we compared isogenic strains with naturally occurring mtrR locus mutations for differences in mtrCDE expression and pump-related phenotypes. Mutations upstream of mtrC, including those within the MtrR binding region and a novel mutation that increases mtrC RNA stability conferred the highest levels of derepression as measured by mtrCDE transcription and resistance to antibiotics, progesterone and antimicrobial peptides. In contrast, mutations within the mtrR coding sequence conferred low to intermediate levels of derepression. In vivo, the mtr mutants were more fit than the wild-type strain, the degree to which paralleled in vitro resistance gradients. These studies establish a hierarchy of mtrR locus mutations with regard to regulation of pump efflux, and suggest selection for more derepressed mutants may occur during mixed infections.

Figure 1. Location of mtr locus mutations used in this study

A schematic of the N. gonorrhoeae mtr locus is shown. The α-helix-encoded region (HTH) of MtrR used for DNA binding is indicated by the hatched pattern. This region is the location of the mutations found in strains DW39, DW45, and MS11. The E202G mutation harbored in strain DW9 is located at the C-terminal end of the MtrR protein, which is hypothesized to be involved in the dimerization of MtrR to itself. The mtrR and mtrCDE transcriptional start sites are indicated by the arrows and are within the intergenic region that contains the MtR binding site and the 13 bp inverted repeat, which is the region where the mutation in strain KH15 is found (Hagman et al., 1995b). The mtr₁₂₀ mutation occurs further upstream of the DNA binding region and is present in strains MS11 and DW120. This mutation has not been described previously, and the G to A change is shown in the detailed DNA sequence.

Figure 2. Expression of MtrE by wild type and mtr mutant bacteria

Outer membrane proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane. The 53-kDa MtrE protein was detected by a rabbit MtrE-specific polyclonal antibody described in the Materials and Methods. With the exception of mtrE mutant DW3, protein samples were loaded in ascending order of the levels of antibiotic resistance. The 32-kDa porin protein is constitutively expressed in N. gonorrhoeae and was detected by staining the PVDF membrane with amido black to show equal loading of the samples. The intensity of the MtrE band relative to strain FA19 and after normalization to porin is reported for each strain below the blot. Strain DW11, not described here, carries a frame-shift mutation in the mtrR gene, that is predicted to cause a 52 kD truncated MtrR protein (Warner et al., 2007).

Figure 3. Transcriptional analysis of mtrA, mtrR, mtrC, and rmp

Quantitative RT-PCR was used to assess the fold difference in levels of mRNA compared to the wild type FA19 bacteria. One representative experiment of the three biological replicates tested is shown. There were no significant differences between levels of rmp or mtrA mRNA. mtrR levels were highly down-regulated or non-existent in strains JF1 and KH15 as described previously (Hagman et al., 1995b, Shafer et al., 1995); these values have been omitted to preserve the scale of the figure. Numbers in parentheses denote the fold increase in mtrC levels compared to wild type FA19 bacteria. Levels of mtrC expression were compared using a students t-test to evaluate differences between DW39, DW120, FA19_MS11mtr, and KH15 gonococci, which exhibit high levels of mtrC expression, and JF1 bacteria, which demonstrate a low-level increase in MIC and mtrC mRNA. ** Denotes p<0.001. The increase in mtrR transcription shown for strain DW120 was 1.7-fold greater than that of strain FA19, but not statistically significant when the averages from three experiments were compared.

Figure 4. Assessment of RNA decay in strain DW120

(A) Equal quantities of RNA from strain FA19 (top) and DW120 (bottom) were reverse transcribed and used in a PCR reaction to determine if there was a difference in the degradation rate of mtrC message. Samples were taken pre (T0) and post rifampicin treatment (+Rif); a second set of cultures to which no rifampicin was added (-Rif) were grown in parallel. (B) RNA samples were also reverse-transcribed and used in a qRT-PCR protocol to quantify amounts of mtrC and rmp RNA from strains FA19 and DW120. These values were used to calculate the RNA half life for mtrC and rmp. The experiment was performed twice and the results were similar.

Figure 5. Progesterone resistance

Suspensions of wild type and mutant gonococci were quantitatively cultured on GC agar plates supplemented with progesterone (35 μg/ml) or without progesterone and incubated overnight. Results are expressed as 100 × (# CFU on GC agar with progesterone divided by # CFU on GC alone). The average % recovery calculated from three independent experiments is shown; bars represent the standard error. Asterisks indicate a significant difference between DW39 (p < 0.05) or DW120 and KH15 (p < 0.01) (unpaired t test). The p value for FA19_MS11mtr versus wild type FA19 was 0.068.

Figure 6. Resistance to antimicrobial peptides

Bacteria were incubated with LL-37, CRAMP, or no peptides and then quantitatively cultured on GC agar. Results are expressed as the percentage of bacteria recovered from wells without peptide, and the graphs shown are representative of one of two or three experiments. Panels A shows the percent recovery of strain FA19 and mtr mutants incubated in CRAMP-38 (4 μg/ml); panel B shows the percent recovery of FA19Sm^R, FA19_MS11mtr, MS11, and DW3_MS11 after incubation in LL-37 or CRAMP-38 (16 μg/ml).

Figure 7. Differential in vivo fitness of mtr mutants compared to the wild type strain

Defined ratios of FA19 Cm^R and mtr mutant bacteria were inoculated into GC broth or estradiol-treated mice to assess the relative fitness of each strain under in vitro and in vivo conditions. The ratio of strains in each inoculum was used in the competitive index (CI) equation as defined in Experimental Procedures. (A) Mixed suspensions were cultured in GC broth (in vitro competition); recovery over the course of growth is expressed as CI. In vivo competition assays between wild type strain FA19Cm^R and strains (B) DW9, (C) DW39, (D) KH15 and (E) DW120 show different degrees of fitness as measured by CI. Each circle in panels B-E represents the CI from each individual mouse; open circles represent cultures from which no Cm^R wild type bacteria were recovered. The bars represent the geometric mean of the data, and the dotted line delineates a CI value of 1.0. In cases where a strain was no longer recovered, the limit of detection (4 CFU/100 μl of vaginal wash) was used to calculate the competitive index.

Figure 8. In vivo competition of mtr mutants

Defined ratios of mutant KH15 bacteria and Cm^R-marked mtr mutants were inoculated into GC broth or estradiol-treated mice to compare the relative fitness under in vitro and in vivo conditions. The ratio of strains in the inoculum was used in the competitive index (CI) equation defined below. (A) Mixed suspensions were cultured in GC broth (in vitro competition); recovery over the course of growth is expressed as CI. The inset shows the optical density of the liquid cultures over time. In vivo competition between strains KH15 and (B) DW9Cm^R (C) DW45Cm^R and (D) DW39Cm^R are shown. In panels B-D, each circle represents the CI value for each individual mouse, and open circles signify mice from which only strain KH15 was recovered, and closed circles represent mice from which both strains were recovered. The CI of 1.0 is denoted by a dashed line, while the geometric mean of each distribution is represented by a bar. In cases where a strain was no longer recovered, the limit of detection (4 CFU/100 μl of vaginal wash) was used to calculate the competitive index. The CI was defined as: (KH15/mtrRCm^R)_output/(KH15/mtrRCm^R)_input, where mtrRCm^R corresponds to strains DW9Cm^R, DW45Cm^R, or DW39Cm^R.