Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system

Abstract

Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg(2+) induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A.

Fig. 1.

Schematic representation of the pmrCAB genes. No amino acid changes were found in PmrA or PmrC. Amino acid replacements predicted in PmrB are identified with the arrows. No substitutions were found in these proteins of ATCC 19606 ColS, ABRIM ColS, and Ab15/133 or in the colistin-susceptible A. baumannii strains. The main domains in PmrB are identified, specifically, (i) the dimerization/phosphoacceptor domain, with the conserved histidine 228, and (ii) the ATP binding site.

Fig. 2.

Relative expression of the pmrCAB genes in the colistin-resistant and -susceptible strains (expression level of pmrCAB in the ColS strains equal to 1). (A) Ab15/132 ColR compared with Ab15/133 ColS; (B) ATCC 19606 ColR compared with ATCC 19606 ColS; (C) ABRIM ColR compared with ABRIM ColS; (D) means for five clinical ColR strains compared with those for five clinical ColS strains. Values are the means and the standard deviations from three independent experiments.

Fig. 3.

Negative-ion MALDI-TOF mass spectra of lipid A isolated from colistin-resistant and -susceptible A. baumannii isolates. (A) Ab15/133 and Ab15/132; (B) ABRIM ColS and ABRIM ColR; (C) ATCC 19606 ColS and ATCC 19606 ColR; (D) ATCC 19606 ColS ΔpmrB and ATCC 19606 ColR ΔpmrB.

Fig. 4.

Proposed structures of the main molecular lipid A species.

Fig. 5.

Negative-ion MALDI-TOF mass spectra of lipid A isolated from strains grown under high-Mg²⁺ conditions (2 mM magnesium sulfate) and low-Mg²⁺ conditions (2 μM magnesium sulfate). (A) ATCC 19606 ColS wild type; (B) ATCC 19606 ColS ΔpmrB.