Many chromosomal genes modulate MarA-mediated multidrug resistance in Escherichia coli

Abstract

Multidrug resistance (MDR) in clinical isolates of Escherichia coli can be associated with overexpression of marA, a transcription factor that upregulates multidrug efflux and downregulates membrane permeability. Using random transposome mutagenesis, we found that many chromosomal genes and environmental stimuli affected MarA-mediated antibiotic resistance. Seven genes affected resistance mediated by MarA in an antibiotic-specific way; these were mostly genes encoding unrelated enzymes, transporters, and unknown proteins. Other genes affected MarA-mediated resistance to all antibiotics tested. These genes were acrA, acrB, and tolC (which encode the major MarA-regulated multidrug efflux pump AcrAB-TolC), crp, cyaA, hns, and pcnB (four genes involved in global regulation of gene expression), and the unknown gene damX. The last five genes affected MarA-mediated MDR by altering marA expression or MarA function specifically on acrA. These findings demonstrate that MarA-mediated MDR is regulated at multiple levels by different genes and stimuli, which makes it both complex and fine-tuned and interconnects it with global cell regulation and metabolism. Such a regulation could contribute to the adaptation and spread of MDR strains and may be targeted to treat antibiotic-resistant E. coli and related pathogens.

FIG. 1.

Complementation of genes whose inactivation reduced MarA-mediated MDR to all antibiotics tested. Those genes whose inactivation reduced MarA-mediated MDR to all antibiotics tested (Table 3) were added back on a plasmid to their respective mutants to study if MarA-mediated MDR was restored. All plasmids were derivatives of the control plasmid pNTR-SD (Table 1). The percentage of MarA-mediated MDR was calculated as explained in Table 2, footnote b, and Table 3, footnote a, with 100% MarA-mediated antibiotic resistance defined as the ratio resulting from dividing, for each antibiotic, the MIC for the ΔmarR parental strain CR1000 bearing the pNTR-SD plasmid by the MIC for the ΔmarRA parental strain CR2000 bearing pNTR-SD. The results are presented as the average ± the standard error of the mean (n = 4). Statistically significant differences are shown as * (P < 0.05) or ** (P < 0.01).

FIG. 2.

Gene expression in mutants with reduced MarA-mediated MDR to all antibiotics tested. The effect of each inactivated gene on the expression of marA, acrA, tolC, micF, and gapA was measured by RT-qPCR (see Materials and Methods). The results are presented as the average ± the standard error of the mean (n = 3 to 5). Statistically significant differences for a mutant compared to the level for the parental strain are shown as * (P < 0.05) or ** (P < 0.01). (A) Fold change in marA expression in different ΔmarR-derivative mutants relative to the level for the ΔmarR parental strain CR1000. Fold 1 means no change in marA expression in a mutant compared to the level for the parental strain. gapA was used as an endogenous reference gene, and its expression was not affected by marA (gapA expression was the same in the ΔmarR and ΔmarRA parental strains; data not shown). Except for pcnB, none of the inactivated genes tested significantly affected the expression of gapA. The pcnB mutant showed reductions in both gapA and marA levels. However, such reductions seem to be an effect specific for these genes and not a general effect produced by pcnB inactivation on all RNAs, since inactivation of pcnB in the ΔmarRA background produced the same reduction in gapA levels but did not decrease the levels of the other genes studied (acrA, tolC, and micF) (data not shown). (B) Effect of inactivation of each gene on MarA induction of acrA, tolC, and micF expression. The percentage of MarA induction of gene expression for each mutant was calculated by comparing the ΔmarR/ΔmarRA ratio (in terms of the number of tran-scripts per ng of RNA) observed for the mutant to that observed for the parental strain, as explained for the percentage of MarA-mediated MDR in Table 3, footnote a.

FIG. 3.

Effect of pcnB inactivation on marA mRNA stability. The absolute numbers of marA transcripts per ng of total RNA before addition of rifampin (time zero) were 2.3·10⁵ for the ΔmarR parental strain (shown in blue), 1.1·10⁵ for the ΔmarR pcnB mutant (shown in red), and 6.3·10³ for the wild-type strain BW25113 (not shown). The half-life of marA mRNA was calculated as detailed in Materials and Methods, using the linear part of the ΔmarR parental strain and ΔmarR pcnB mutant curves (shown as a continuous line; data within the first minute). No significant differences were found: the marA mRNA half-lives were 0.21 min for the ΔmarR parental strain and 0.22 min for its derivative pcnB mutant.

FIG. 4.

Effects of crp, cyaA, and hns on adaptation of MarA-mediated MDR to environmental stimuli. The percentage of MarA-mediated MDR was calculated as explained in Table 3, footnote a. The results are presented as the average ± the standard error of the mean (n = 3). When different conditions for the same strains were compared, addition of 0.4% glucose or 300 mM NaCl was found to produce statistically significant differences in MarA-mediated MDR in the parental strain (P was <0.01 for all antibiotics except for cefoxitin, in which case P was <0.05 for NaCl and P was <0.1 for glucose; these statistical significances are shown in the figure as “**,” “*,” and “+,” respectively), whereas these additions produced no differences in the mutants studied. Levels of MarA-mediated MDR were significantly different between the parental strain and the mutants in the absence of glucose and NaCl (P < 0.01 [see Table 3]; these statistical significances are not indicated in this figure), but no significant differences between them were found in the presence of glucose or NaCl.

FIG. 5.

Regulation of MarA-mediated MDR. The figure was produced using data from the literature (see main text) and the results obtained here. Functional interactions, either direct or indirect, are represented as arrows for activation/induction and as “⊣” for repression. New genes found here to affect MarA-mediated MDR are in blue.