The MerR-like transcriptional regulator BrlR contributes to Pseudomonas aeruginosa biofilm tolerance

Abstract

Biofilms are composed of surface-attached microbial communities. A hallmark of biofilms is their profound tolerance of antimicrobial agents. While biofilm drug tolerance has been considered to be multifactorial, our findings indicate, instead, that bacteria within biofilms employ a classical regulatory mechanism to resist the action of antimicrobial agents. Here we report that the transcriptional regulator BrlR, a member of the MerR family of multidrug transport activators, plays a role in the high-level drug tolerance of biofilms formed by Pseudomonas aeruginosa. Expression of brlR was found to be biofilm specific, with brlR inactivation not affecting biofilm formation, motility, or pslA expression but increasing ndvB expression. Inactivation of brlR rendered biofilms but not planktonic cells grown to exponential or stationary phase significantly more susceptible to hydrogen peroxide and five different classes of antibiotics by affecting the MICs and the recalcitrance of biofilms to killing by microbicidal antimicrobial agents. In contrast, overexpression of brlR rendered both biofilms and planktonic cells more tolerant to the same compounds. brlR expression in three cystic fibrosis (CF) isolates was elevated regardless of the mode of growth, suggesting a selection for constitutive brlR expression upon in vivo biofilm formation associated with chronic infections. Despite increased brlR expression, however, isolate CF1-8 was as susceptible to tobramycin as was a ΔbrlR mutant because of a nonsense mutation in brlR. Our results indicate for the first time that biofilms employ a specific regulatory mechanism to resist the action of antimicrobial agents in a BrlR-dependent manner which affects MIC and recalcitrance to killing by microbicidal antimicrobial agents.

FIG 1

BrlR is a member of the MerR family of transcriptional regulator functioning as multidrug transporter activators. (A) brlR transcripts are not detected in planktonic cells grown to the exponential (PE) and stationary (PS) phases but are present in biofilms of various ages (B3d, 3-day biofilm; B6d, 6-day biofilm). mreB was used as a control. Transcript abundance was visualized after 30 cycles by separating the transcripts on a 1% agarose gel. (B) BrlR domain organization. HTH_BmrR, helix-turn-helix DNA binding domain of the MerR BmrR transcription regulator; GyrI-like, GyrI-like small-molecule binding domain; AA, amino acid position. (C) Alignment of BrlR (BrlR_Pa_) with other known MerR-like transcriptional regulators, including TipA from S. lividans (TipA_Sl_), MtaN from B. subtilis (MtaN_Bs_), and BmrR from B. subtilis (BmrR_Bs_). Colons denote residues with strong conservation (score of >0.5 in PAM250 matrix), and periods denote residues with weak conservation (score of ≤0.5). Asterisks denote identical amino acids. Arrows denote residues with side chains contributing to the core of the DNA binding protein (29).

FIG 2

Inactivation of brlR does not alter the susceptibility of planktonic cells to antimicrobial agents. Shown are the susceptibilities of planktonic cells grown to the exponential (A, C) or stationary (B, D) phase to 50 μg/ml tobramycin (A, B) and 0.3% hydrogen peroxide (C, D). Planktonic cells were treated for 30 min with hydrogen peroxide and for 1 h with tobramycin. **, significantly different (P < 0.001) from PAO1 as indicated by ANOVA and SigmaStat.

FIG 3

Inactivation of brlR does not alter P. aeruginosa architecture or colony morphology or pslA but does affect ndvB expression. (A) Biofilm architecture of P. aeruginosa PAO1 strains with brlR inactivation or overexpression. Biofilms were stained with the LIVE/DEAD Bac Light viability stain (Invitrogen Corp.). Bars = 100 μm. Inactivation of brlR has no effect on colony morphology (B) or pslA expression (C). (D) Increased ndvB expression in strains with brlR inactivated.

FIG 4

BrlR contributes to drug tolerance of biofilm cells. (A) Inactivation of brlR resulted in 1-day biofilms with significantly increased susceptibility to tobramycin (100 μg/ml). Overexpression of brlR restored their susceptibility to tobramycin to the wild-type level. (B) Inactivation of brlR resulted in 1-day biofilms with significantly increased their susceptibility to 0.3% hydrogen peroxide. Expression of brlR in the ΔbrlR mutant restored its susceptibility to hydrogen peroxide to the wild-type level. (C) Increased susceptibility of the ΔbrlR mutant biofilm formed after 1 day was observed with tetracycline (Tet; 100 μg/ml), kanamycin (Km; 150 μg/ml), norfloxacin (Nor; 450 μg/ml), and trimethoprim (Trim; 150 μg/ml). Biofilms were treated for 1 h. **, significantly different (P < 0.001) from PAO1 as indicated by ANOVA and SigmaStat. *, significantly different (P < 0.001) from PAO1 as indicated by t test.

FIG 5

BrlR regulates resistance to killing in P. aeruginosa biofilms. P. aeruginosa PAO1 and ΔbrlR mutant biofilms were grown for 3 days and subsequently treated for 24 h under continuous flowing conditions before the recovery and enumeration of surviving cells. (A) Biofilm susceptibility to tobramycin as determined by viable CFU counts. CFU counts were obtained from biofilm tube reactors having an inner surface area of 25 cm². The number of viable ΔbrlR mutant cells was below the detection limit at the highest concentrations of tobramycin tested. (B) Biofilm susceptibility to tobramycin as determined by log reduction. Total killing of ΔbrlR mutant biofilm cells was achieved at 40 μg/ml of tobramycin. In contrast, P. aeruginosa PAO1 biofilms maintained a steady level of persisting survivors at concentrations higher than 40 μg/ml of tobramycin. (C, D) Biofilm susceptibility to norfloxacin was determined similarly. Error bars denote standard deviations. (E) Inactivation of brlR eliminates resistance to killing of P. aeruginosa biofilms. Biofilms (3 days old) formed by wild-type PAO1 and the ΔbrlR mutant were treated with 50 μg/ml of tobramycin under continuous flowing conditions for 24 h and then allowed to recover under flowing conditions in the absence of tobramycin for 24 h. Viable cells were collected and enumerated after the recovery period. CFU counts were obtained from biofilm tube reactors having an inner surface area of 25 cm². Lines indicate the difference between the highest and lowest numbers of viable cells recovered, while bars represent the distribution between the mean and median of the replicates. **, significantly different (P < 0.001) from untreated biofilms. (F) Biofilms formed after 5 days by wild-type PAO1, PAO1/pMJT1, the ΔbrlR mutant, and the overexpresser PAO1/pMJT-brlR-V5/6×His were treated with tobramycin under continuous flow for 24 h. The effect of tobramycin was examined using LIVE/DEAD Bac Light viability stain. Images for PAO1 and the ΔbrlR mutant were acquired with the same microscope settings. The percentages of the biofilm population stained with PI prior to and following treatment with tobramycin were quantitated using COMSTAT. **, significantly different (P < 0.001) from PAO1 as indicated by ANOVA and SigmaStat. White bars, 100 μm.

FIG 6

While brlR is overexpressed in clinical CF isolates under both planktonic and biofilm growth conditions, isolate CF1-8 is more susceptible to tobramycin under biofilm growth conditions. (A) brlR transcript abundance was determined by qRT-PCR in clinical isolates grown as biofilms for 3 days and in planktonic culture. Transcript levels were normalized to PAO1. (B) Susceptibility determination of P. aeruginosa PAO1 and clinical CF isolates grown planktonically and as biofilms (1 day). Planktonic cells were treated with 50 μg/ml tobramycin for 1 h, while biofilms were treated with 100 μg/ml tobramycin for 1 h under flowing conditions. **, significantly different (P < 0.001) from PAO1 as indicated by ANOVA and SigmaStat.

FIG 7

Isolate CF1-8 harbors a nonsense mutation in BrlR. (A) Alignment of BrlR sequences of PAO1, CF1-8, and CF1-13. CF1-8 harbors a point mutation resulting in a premature stop codon at amino acid position 88. CF1-13 harbors a mutation resulting in an amino acid substitution (Y→C). The arrow indicates the location of the N-terminal DNA binding domain of BrlR. (B) Complementation of the ΔbrlR mutant with the N-terminal DNA binding site of BrlR alone (BrlR-N) does not restore the susceptibility to tobramycin of this strain to the wild-type level. (C) Inactivation of brlR in isolate CF1-13 increases the susceptibility of this strain to tobramycin, while inactivation of brlR in isolate CF1-8 has no effect. Strains were grown planktonically to the exponential phase and treated with 50 μg/ml tobramycin for 1 h. *, significantly different (P < 0.001) from CF1-13 as indicated by t test.