EnvR is a potent repressor of acrAB transcription in Salmonella

Abstract

Background: Resistance nodulation division (RND) family efflux pumps, including the major pump AcrAB-TolC, are important mediators of intrinsic and evolved antibiotic resistance. Expression of these pumps is carefully controlled by a network of regulators that respond to different environmental cues. EnvR is a TetR family transcriptional regulator encoded upstream of the RND efflux pump acrEF.

Methods: Binding of EnvR protein upstream of acrAB was determined by electrophoretic mobility shift assays and the phenotypic consequence of envR overexpression on antimicrobial susceptibility, biofilm motility and invasion of eukaryotic cells in vitro was measured. Additionally, the global transcriptome of clinical Salmonella isolates overexpressing envR was determined by RNA-Seq.

Results: EnvR bound to the promoter region upstream of the genes coding for the major efflux pump AcrAB in Salmonella, inhibiting transcription and preventing production of AcrAB protein. The phenotype conferred by overexpression of envR mimicked deletion of acrB as it conferred multidrug susceptibility, decreased motility and decreased invasion into intestinal cells in vitro. Importantly, we demonstrate the clinical relevance of this regulatory mechanism because RNA-Seq revealed that a drug-susceptible clinical isolate of Salmonella had low acrB expression even though expression of its major regulator RamA was very high; this was caused by very high EnvR expression.

Conclusions: In summary, we show that EnvR is a potent repressor of acrAB transcription in Salmonella, and can override binding by RamA so preventing MDR to clinically useful drugs. Finding novel tools to increase EnvR expression may form the basis of a new way to prevent or treat MDR infections.

Figure 1.

EnvR binds the promoter of acrAB of Salmonella SL1344. Purified EnvR protein (0.8 mg/mL) binds the promoter of acrAB, as shown by EMSA. The promoter of acrAB was purified and diluted to a final concentration range of 15–1.5 ng/µL. The binding of EnvR to this promoter resulted in the shift of the fluorescent band, indicating the formation of a DNA:EnvR complex. The first dilution (1:10; 15 ng/µL) was then selected as the standard DNA concentration in further EMSA experiments.

Figure 2.

Confirmation of the binding site of Salmonella EnvR. (a) The promoter of acrAB was synthesized containing the following mutations in the known palindromic binding site of AcrR: T29G, C31T, T33G, C35A, T37C, A44G, G46T, A48C, G50A, A52C. (b) The binding of EnvR to the WT or mutated promoter (acrAB_mut) of acrAB as determined by EMSA. The binding of EnvR to the promoter of acrAB was abolished when these mutations were present. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Figure 3.

Overexpression of EnvR reduces transcription of acrAB and AcrB protein level. (a) Expression of acrA and acrB measured by RT–PCR in SL1344 and a strain overexpressing envR. Data shown are the mean of three independent biological replicates ± SE. (b) Western blot for AcrB showing decreased protein level when envR is overexpressed.

Figure 4.

Phenotypic effect of inactivation or overexpression of envR in Salmonella SL1344. (a) Accumulation of Hoechst 33342 following inactivation or overexpression of envR. Data shown are the mean of three independent biological replicates ± SEM. (b) Swimming motility. Data shown are the mean ± SEM of three independent biological repeats. (c) Invasion of Salmonella strains into human intestinal cells (INT-407). Data shown are the mean ± SEM of three independent biological repeats. ns, not significant; ***P < 0.001.

Figure 5.

Effect of envR deletion or overexpression in different Salmonella backgrounds. (a) Fold change in gene expression acrA relative to SL1344 measured by real-time RT–PCR. (b) Western blot for AcrB. *P < 0.05; ***P < 0.001.