The ABC of Biofilm Drug Tolerance: the MerR-Like Regulator BrlR Is an Activator of ABC Transport Systems, with PA1874-77 Contributing to the Tolerance of Pseudomonas aeruginosa Biofilms to Tobramycin

Abstract

A hallmark of biofilms is their tolerance to killing by antimicrobial agents. In Pseudomonas aeruginosa, biofilm drug tolerance requires the c-di-GMP-responsive MerR transcriptional regulator BrlR. However, the mechanism by which BrlR mediates biofilm drug tolerance has not been elucidated. Here, we demonstrate that BrlR activates the expression of at least 7 ABC transport systems, including the PA1874-PA1875-PA1876-PA1877 (PA1874-77) operon, with chromatin immunoprecipitation and DNA binding assays confirming BrlR binding to the promoter region of PA1874-77. Insertional inactivation of the 7 ABC transport systems rendered P. aeruginosa PAO1 biofilms susceptible to tobramycin or norfloxacin. Susceptibility was linked to drug accumulation, with BrlR contributing to norfloxacin accumulation in a manner dependent on multidrug efflux pumps and the PA1874-77 ABC transport system. Inactivation of the respective ABC transport system, furthermore, eliminated the recalcitrance of biofilms to killing by tobramycin but not norfloxacin, indicating that drug accumulation is not linked to biofilm drug tolerance. Our findings indicate for the first time that BrlR, a MerR-type transcriptional activator, activates genes encoding several ABC transport systems, in addition to multiple multidrug efflux pump genes. Moreover, our data confirm a BrlR target contributing to drug tolerance, likely countering the prevailing dogma that biofilm tolerance arises from a multiplicity of factors.

Keywords: ABC transport system; ABC transporter; MBC biofilm assay; MexAB-OprM; PA4142-44; PA5503-05; biofilm MBC; biofilm drug tolerance; drug accumulation; kdpFABC; opuC; phosphotransfer.

FIG 1

Intracellular accumulation of norfloxacin in biofilm cells requires the presence of BrlR, the MexAB-OprM and MexEF-OprN efflux pumps, and the PA1874-77 ABC transport system. Biofilms were grown for 3 days and subsequently exposed to norfloxacin (450 μg/ml) for 24 h. Lysates were subsequently analyzed using antibiotic accumulation assays. Lysates were obtained from biofilms formed by wild-type strains PAO1 and by mutant strains in which brlR was inactivated or that overexpressed brlR (A), PAO255 mutant strains in which mexAB-oprM and mexEF-oprN were inactivated and that overexpressed brlR or mexAB-oprM and mexEF-oprN (B), and mutant strains in which PA1876 was inactivated and the PA1876::IS mutant overexpressed brlR or PA1875-77 (C). Representative images above the graphs show the zone of clearing in response to lysates obtained from biofilms. The diameter of the zone of clearing (in millimeters), indicative of intracellular antibiotic accumulation, was determined at 24 h of incubation. Experiments were performed in triplicate, and error bars represent standard deviations. The genotype of PAO255 is ΔmexAB-oprM ΔmexEF-oprN. *, the values are significantly different from the value for PAO1 or PAO1/pJN105 (P < 0.1); **, the values are significantly different from the value for PAO1 or PAO1/pJN105 (P < 0.01).

FIG 2

BrlR activates the expression of PA1874 and PA1875 and binds to the promoter region of PA1874-77. (A) Fold change in the abundance of the PA1874 and PA1875 transcripts in biofilms formed by the ΔbrlR mutant and PAO1/pJN-brlR relative to the abundance in wild-type biofilms, as determined using qRT-PCR. The mreB housekeeping gene was used for the normalization of transcript abundance. (B) Fold change in the abundance of the PA1874, PA1875, and PA1877 transcripts in the ΔbrlR mutant and PAO1/pMJT-brlR grown planktonically to exponential phase relative to that in wild-type strain PAO1. (C) Fold enrichment of the promoter DNA of PA1874, brlR, and pscEF in samples with BrlR-V5/His₆ submitted to ChIP compared to control samples (for which ChIP was carried out in the presence of untagged BrlR), as determined by qPCR. P_pscEF was used as a negative control, while enrichment of the brlR promoter region (P_brlR) was used as a positive control. (D) Streptavidin (SA) magnetic bead binding assay demonstrating tagged BrlR binding to the biotinylated PA1874 promoter. BrlR binding to the PA1874 promoter was outcompeted using nonbiotinylated promoter DNA (P_PA1874-NB).

FIG 3

Inactivation of the genes in the PA1875-77 operon does not affect the susceptibility of P. aeruginosa cells grown planktonically to antimicrobial agents. (A) Average number of viable cells, expressed as the number of CFU, detectable in exponential-phase cultures of P. aeruginosa PAO1 and strains in which brlR was inactivated or that harbored transposon insertions in the PA1875, PA1876, and PA1877 genes prior to treatment with antibiotics. (B) Susceptibility of P. aeruginosa PAO1 and strains in which brlR was inactivated or that harbored transposon insertions in the PA1875, PA1876, and PA1877 genes to tobramycin (50 μg/ml) and norfloxacin (150 μg/ml). (C and D) Susceptibility of P. aeruginosa PAO1 and strains harboring transposon insertions in PA1875 and PA1876 genes to levofloxacin (10 μg/ml) and prulifloxacin (5 μg/ml). diluent only, P. aeruginosa PAO1 biofilms were exposed to the diluent used to resuspend prulifloxacin. All strains were grown planktonically to exponential phase prior to treatment with the antibiotics for 30 min at 37°C. Susceptibility is expressed as the reduction in the log₁₀ number of CFU. All experiments were carried out at least in triplicate. Error bars indicate standard deviations.

FIG 4

Components of the PA1874-77 ABC transport system contribute to the heightened resistance of P. aeruginosa PAO1 biofilms to tobramycin, norfloxacin, and prulifloxacin but not levofloxacin under continuous-flow conditions. The indicated strains were grown as biofilms under continuous-flow conditions for 3 days and subsequently exposed to antibiotics for 1 h under flowing conditions. Then, the biofilms were harvested, homogenized, serially diluted, and spread plated onto LB agar. Viability was determined via measurement of the number of CFU. Susceptibility was expressed as the reduction in the log₁₀ number of CFU. Biofilms formed by the P. aeruginosa wild-type strains PA14 and PAO1 were used as controls. (A) Susceptibility of the biofilms formed by P. aeruginosa PA14 and the isogenic mutant strain in which PA1874-77 was inactivated (ΔPA1874-PA1877) to tobramycin (150 μg/ml). (B) Susceptibility of biofilms formed by the indicated strains to tobramycin (150 μg/ml) and norfloxacin (450 μg/ml). (C and D) Susceptibility of biofilms formed by the indicated strains to prulifloxacin (10 μg/ml) (C) and increasing concentrations of levofloxacin (5, 20, and 50 μg/ml) (D). (E) Susceptibility of biofilms formed by PAO1 and the PA1875::IS and PA1876::IS mutant strains overexpressing PA1875-77 to tobramycin (150 μg/ml) and norfloxacin (450 μg/ml). Biofilms formed by P. aeruginosa PAO1 harboring the empty pJN105 plasmid were used as controls. (F) Susceptibility of biofilms formed by the PA1875::IS, PA1876::IS, and PA1877::IS mutant strains overexpressing brlR to tobramycin (150 μg/ml) and norfloxacin (450 μg/ml). Biofilms formed by P. aeruginosa PAO1 harboring the empty pJN105 plasmid were used as controls. All experiments were carried out at least in triplicate. Error bars indicate standard deviations. *, the value is significantly different from the value for PAO1 (P < 0.001).

FIG 5

Biofilms formed by cells in which the PA1875-77 operon was inactivated demonstrate a biofilm architecture altered from that of wild-type biofilms. (A) Representative confocal images showing the architecture of biofilms formed by P. aeruginosa PAO1 and strains harboring transposon insertions in the PA1875, PA1876, and PA1877 genes. Biofilms were grown for 5 days under continuous-flow conditions in flow cells and stained using Live/Dead BacLight viability stain (Life Technologies Corp.) prior to confocal microscopy. Representative images are shown. Bars = 100 μm. (B and C) Confocal images were subjected to quantitative analysis using the Comstat program to determine the biofilm biomass (B) and the average and maximum biofilm thickness (C). (D) Average number of viable cells, expressed as the log₁₀ number of CFU, detectable in biofilms formed by P. aeruginosa PAO1 and strains harboring transposon insertions in the PA1875, PA1876, and PA1877 genes. All experiments were carried out at least in triplicate. Error bars indicate standard deviations. *, the value is significantly different from the value for PAO1 (P < 0.001).

FIG 6

Inactivation of PA1876 eliminates the recalcitrance of P. aeruginosa biofilms to killing by tobramycin but not norfloxacin, as determined using biofilm MBC assays. (A and B) P. aeruginosa PAO1, ΔbrlR, and PA1876::IS were grown as biofilms for 3 days and subsequently treated for 24 h with norfloxacin (50, 100, 200, and 450 μg/ml) (A) or tobramycin (40, 75 and 150 μg/ml) (B) under continuous-flow conditions. (C) Biofilms formed by the PA1876::IS mutant overexpressing brlR (PA1876::IS/pJN-brlR) were treated for 24 h with increasing concentrations of tobramycin (0 to 600 μg/ml). The PA1876::IS mutant harboring the empty pJN105 vector was used as a control. Following 24 h of treatment, biofilms were recovered from the tube reactor and the surviving cells were enumerated by determination of viable counts. Biofilm CFU, number of viable cells obtained from the biofilm tube reactor having an inner surface area of 25 cm². #, no viable bacteria were detected. All experiments were carried out in triplicate. Error bars denote standard deviations.

FIG 7

BrlR regulates several ABC transport and efflux systems to confer biofilm resistance. (A) Abundance of the transcripts of genes encoding efflux and ABC transport systems in biofilms formed by the ΔbrlR mutant and PAO1/pJN-brlR relative to that in wild-type biofilms. The mreB housekeeping gene was used for the normalization of transcript abundance. (B) Susceptibility of 3-day-old biofilms formed by the wild type and strains in which brlR was inactivated or that harbored transposon insertions (PA1633::IS, PA3402::IS, PA3836::IS, PA3865::IS, PA3888::IS, PA4142::IS, and PA5503::IS) to tobramycin (150 μg/ml) and norfloxacin (450 μg/ml). Biofilms were treated for 1 h under flowing conditions. Susceptibility was recorded as the reduction in the log₁₀ number of CFU. Error bars represent standard deviations. *, the value is significantly different (P < 0.001) from the value for PAO1.