PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients

Abstract

Pseudomonas aeruginosa can develop resistance to polymyxin and other cationic antimicrobial peptides. Previous work has shown that mutations in the PmrAB and PhoPQ regulatory systems can confer low to moderate levels of polymyxin resistance (MICs of 8 to 64 mg/liter) in laboratory and clinical strains of this organism. To explore the role of PhoPQ in high-level clinical polymyxin resistance, P. aeruginosa strains with colistin MICs > 512 mg/liter that had been isolated from cystic fibrosis patients treated with inhaled colistin (polymyxin E) were analyzed. Probable loss-of-function phoQ alleles found in these cystic fibrosis strains conferred resistance to polymyxin. Partial and complete suppressor mutations in phoP were identified in some cystic fibrosis strains with resistance-conferring phoQ mutations, suggesting that additional loci can be involved in polymyxin resistance in P. aeruginosa. Disruption of chromosomal phoQ in the presence of an intact phoP allele stimulated 4-amino-l-arabinose addition to lipid A and induced transcription from the promoter of the pmrH (arnB) operon, consistent with the known role of this lipid A modification in polymyxin resistance. These results indicate that phoQ loss-of-function mutations can contribute to high-level polymyxin resistance in clinical strains of P. aeruginosa.

Fig. 1.

Effect of phoPQ mutations on P. aeruginosa Pm resistance. (A) PMB quantitative bactericidal assay of strains 1995 (PAK ΔpmrAB phoQ6) and 2248 (PAK ΔpmrAB ΔphoPQ). (B) Alternative PMB plate assay of the PAK ΔphoPQ strain expressing the phoPQ⁺, phoPQ6, or phoP⁺ allele inserted in plasmid pJN105D. (C) Alternative PMB plate assay of chromosomal deletion mutants PAK ΔphoQ and PAK ΔpmrAB ΔphoQ as well as PAK ΔphoQ expressing the phoPQ⁺ alleles inserted in plasmid pJN105D. (D) Alternative PMB plate assay of the PAK ΔphoPQ strain expressing mutant phoPQ alleles from CF clinical isolates in plasmid pJN105D. The phoP⁺Q21 and phoP⁺Q24 alleles encode deletions from PhoQ of 2 and 276 amino acids, respectively, while the phoP24Q24 allele also encodes replacement in PhoP of Arg 118 with Cys.

Fig. 2.

Lipid A structures of Pm^s and Pm^r strains. (A) Diagram of lipid A structural modifications found in P. aeruginosa. (B to D) MALDI-TOF mass spectra for (B) PAK ΔpmrAB (strain 1812), (C) PAK ΔpmrAB phoQ6 (strain 1995), and (D) PAK ΔpmrAB ΔphoPQ (strain 2244).

Fig. 3.

Transcriptional activity of phoPQ⁺, phoPQ6, phoP⁺, phoP⁺Q21, phoP23Q23, phoP23, phoP24Q24, phoP24, and phoP⁺Q24 alleles expressed from pJN105D in reporter strain PAO1 ΔphoPQ Ω[attP::Φ(P_pmrH-lacZ⁺)]. Data shown represent the means of the results of triplicate biological experiments; error bars represent ± standard deviations. Fold induction values shown were calculated relative to the values determined for the WT phoPQ⁺ allele.