A PmrB-Regulated Deacetylase Required for Lipid A Modification and Polymyxin Resistance in Acinetobacter baumannii

Abstract

Emerging resistance to "last-resort" polymyxin antibiotics in Gram-negative bacteria is a significant threat to public health. We identified the Acinetobacter baumannii NaxD deacetylase as a critical mediator of lipid A modification resulting in polymyxin resistance and demonstrated that naxD is regulated by the sensor kinase PmrB. This represents the first description of a specific PmrB-regulated gene contributing to polymyxin resistance in A. baumannii and highlights NaxD as a putative drug target to reverse polymyxin resistance.

FIG 1

PmrB-dependent overexpression of naxD in a polymyxin-resistant strain of A. baumannii. The wild-type polymyxin-sensitive strain 17978 (WT), the polymyxin-resistant derivative R2, the isogenic R2 pmrB deletion mutant, and the pmrB-complemented (R2 pmrB comp) strains were grown in lysogeny broth (LB) supplemented with 1 mM MgCl₂ to an optical density at 600 nm of 0.6. RNA was harvested and used for quantitative real-time PCR analysis of naxD expression in relation to the housekeeping 16S rRNA. **, P < 0.005; ***, P < 0.0005.

FIG 2

NaxD is required for the galactosamine modification of A. baumannii lipid A. (A) Quantification of lipids in the wild-type polymyxin-susceptible A. baumannii 17978 (WT) strain, the polymyxin-resistant derivative R2, the isogenic naxD deletion mutant strain (R2ΔnaxD), and the naxD-complemented (R2 ΔnaxD^comp) strain. (B to D) The peaks corresponding to bis-phosphorylated hepta-acylated lipid A (m/z 1,910) (B), to that with phosphoethanolamine (m/z 2,033) (C), and to that with galactosamine (m/z 2,072) (D) are present in R2, but the galactosamine is absent in the R2ΔnaxD mutant. For each spectrum, the m/z range between 1,600 and 2,300 is shown. Note that phosphate additions can be at either the 1 or the 4′ position. Ion peaks m/z 1,728 and m/z 1,882 correspond to bis-phosphorylated hepta-acylated lipid A (m/z 1,910) with the loss of a C₁₂ fatty acid and C₂H₄, respectively.

FIG 3

A. baumannii NaxD is required for polymyxin resistance. The A. baumannii wild-type strain, polymyxin-susceptible A. baumannii strain 17978 (WT), polymyxin-resistant strain R2, the R2ΔnaxD mutant, and the naxD-complemented strain (R2ΔnaxD comp) were incubated with 7 μg/ml polymyxin B or 10 μg/ml colistin for 2 h, and CFU were enumerated after plating. **, P < 0.005; ***, P < 0.0005.

FIG 4

Model of the role of A. baumannii NaxD in resistance to polymyxins. Schematics of polymyxin-resistant wild-type A. baumannii and naxD (ΔnaxD) mutant bacteria are shown. (1) PmrB phosphorylates and activates PmrA upon sensing of environmental stimuli (or when rendered constitutively active due to an amino acid mutation, often in the kinase domain). PmrA subsequently binds the naxD promoter, facilitating transcription. (2) NaxD deacetylates N-acetylgalactosamine (GalNAc) to galactosamine (GalN), which is attached to a lipid carrier and transported from the cytosolic to the periplasmic side of the inner membrane. GalN is subsequently added to the 1 position phosphate of lipid A. The lipid A is also modified with phosphoethanolamine (pEtN) through a NaxD-independent process. (3) Lipopolysaccharide (LPS), composed of lipid A, core, and O antigen, is then transported to the outer leaflet of the outer membrane. (4) GalN and pEtN are positively charged, and these modifications to lipid A increase the surface charge of the bacteria such that positively charged polymyxin antibiotics (pentagons) are repelled. (5) In naxD mutant bacteria, LPS lacks the lipid A GalN modification. (6) Therefore, the bacterial surface charge is decreased, allowing polymyxins to interact with and disrupt the bacterial membranes.