Adaptive resistance to the "last hope" antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS

Abstract

As multidrug resistance increases alarmingly, polymyxin B and colistin are increasingly being used in the clinic to treat serious Pseudomonas aeruginosa infections. In this opportunistic pathogen, subinhibitory levels of polymyxins and certain antimicrobial peptides induce resistance toward higher, otherwise lethal, levels of these antimicrobial agents. It is known that the modification of lipid A of lipopolysaccharide (LPS) is a key component of this adaptive peptide resistance, but to date, the regulatory mechanism underlying peptide regulation in P. aeruginosa has remained elusive. The PhoP-PhoQ and PmrA-PmrB two-component systems, which control this modification under low-Mg2+ conditions, do not appear to play a major role in peptide-mediated adaptive resistance, unlike in Salmonella where PhoQ is a peptide sensor. Here we describe the identification and characterization of a novel P. aeruginosa two-component regulator affecting polymyxin-adaptive resistance, ParR-ParS (PA1799-PA1798). This system was required for activation of the arnBCADTEF LPS modification operon in the presence of subinhibitory concentrations of polymyxin, colistin, or the bovine peptide indolicidin, leading to increased resistance to various polycationic antibiotics, including aminoglycosides. This study highlights the complexity of the regulatory network controlling resistance to cationic antibiotics and host peptides in P. aeruginosa, which has major relevance in the development and deployment of cationic antimicrobials.

FIG. 1.

Identification of the parRS operon. (A) Fold induction of pPA3552::lux fusion in BM2-glucose minimal medium supplemented with 2 mM Mg²⁺ in the presence of 4 μg/ml indolicidin compared to induction in the absence of indolicidin in the WT (H103) and mutant strains UW-PA1798 (UW-1798) and UW-PA1799 (UW-1799). The pPA3552::lux fusion contained a promoterless lux cassette fused to the promoter of the arnB gene and reported on the transcription of the eight-gene operon encoding the arn LPS modification operon. The results provided correspond to one representative experiment of at least three independent assays which displayed the same trends. (B) Schematic representation showing the genomic context of the parRS operon. The black arrows represent ORFs.

FIG. 2.

ParR-ParS mediates the upregulation of the LPS modification operon in response to certain antimicrobial peptides. (A) Induction of pPA3552::lux fusion in bacteria grown in BM2-glucose minimal medium supplemented with 20 μM Mg²⁺, 2 mM Mg²⁺, or 2 mM Mg²⁺ and 4 μg/ml indolicidin. The WT (H103), parR and parS mutants, and the complemented parR mutant were studied. The pPA3552::lux fusion contained a promoterless lux cassette fused to the promoter of the arnB gene and reported on the transcription of the eight-gene operon encoding the arn LPS modification operon. Luminescence is shown in relative light units per optical density (RLU/OD). The results provided correspond to one representative experiment of at least four independent assays which displayed the same trends. (B) Fold induction of pPA3552::lux fusion in the WT (H103) and the parR mutant grown in BM2-glucose minimal medium supplemented with 2 mM Mg²⁺ and different antimicrobial compounds. The values shown here correspond to the means plus standard deviations (error bars) of four independent experiments at the concentration of each antibiotic leading to the highest induction level. These antibiotics and concentrations were as follows: tobramycin, 0.5 μg/ml; polymyxin B, 1 μg/ml; indolicidin, 4 μg/ml; colistin, 1 μg/ml; CP28, 3 μg/ml; polyphemusin, 2 μg/ml; and LL-37, 32 μg/ml.

FIG. 3.

Comparison of intrinsic (A) and adaptive (B) polymyxin B resistance in the WT and parR mutant. Sensitivity was analyzed by means of killing curves. Cells from the different strains were grown to mid-log phase in BM2-glucose minimal medium with 2 mM Mg²⁺ (A) or the same medium supplemented with 4 μg/ml indolicidin (B) and exposed to 1 μg/ml (A) or 10 μg/ml (B) of polymyxin B. Diluted aliquots were then plated to assay for survivors. The results presented correspond to one representative experiment for each condition out of four independent experiments which produced identical trends.