The Pseudomonas aeruginosa lipid A deacylase: selection for expression and loss within the cystic fibrosis airway

Abstract

Lipopolysaccharide (LPS) is the major surface component of gram-negative bacteria, and a component of LPS, lipid A, is recognized by the innate immune system through the Toll-like receptor 4/MD-2 complex. Pseudomonas aeruginosa, an environmental gram-negative bacterium that opportunistically infects the respiratory tracts of patients with cystic fibrosis (CF), can synthesize various structures of lipid A. Lipid A from P. aeruginosa strains isolated from infants with CF has a specific structure that includes the removal of the 3 position 3-OH C10 fatty acid. Here we demonstrate increased expression of the P. aeruginosa lipid A 3-O-deacylase (PagL) in isolates from CF infants compared to that in environmental isolates. PagL activity was increased in environmental isolates by growth in medium limited for magnesium and decreased by growth at low temperature in laboratory-adapted strains of P. aeruginosa. P. aeruginosa PagL was shown to be an outer membrane protein by isopycnic density gradient centrifugation. Heterologous expression of P. aeruginosa pagL in Salmonella enterica serovar Typhimurium and Escherichia coli resulted in removal of the 3-OH C14 fatty acid from lipid A, indicating that P. aeruginosa PagL recognizes either 3-OH C10 or 3-OH C14. Finally, deacylated lipid A species were not observed in some clinical P. aeruginosa isolates from patients with severe pulmonary disease, suggesting that loss of PagL function can occur during long-term adaptation to the CF airway.

FIG. 1.

Proposed pathway for the biosynthesis of P. aeruginosa lipid A structures. For all structures, the molecular weight of the singly charged lipid A species is indicated. *, locations of the C12 and 2-OH C12 fatty acids that may be reversed on the P. aeruginosa lipid A structures. Mol wt, molecular weight.

FIG. 2.

Identification of P. aeruginosa PAO-1 mutant strain that lacked deacylated lipid A species. (A and B) MALDI-TOF MS in the negative-reflection ion mode; a transposon insertion mutant designated 32751 lacked deacylated lipid A species, compared to the laboratory adapted PAO-1 strain. (C and D) MS of lipid A analysis of PAO-1 or 32751 strains containing pPAPagL (pDEST JN105 derivative harboring P. aeruginosa pagL-PA4661). Adjacent peaks that differ by 80 or 16 m/z units represent the loss of a phosphate group or the addition of an additional hydroxyl group, respectively.

FIG. 3.

Complementation of P. aeruginosa PA4661 in trans restoring deacylase activity to null-mutant strain. (A) GC analysis of total fatty acid content of lipid A isolated from the laboratory-adapted wild-type PAO-1 (white bars) or 32751 (filled bars) strains is shown. (B) GC analysis of total fatty acid content of lipid A isolated from PAO-1 (white bars) or 32751 (filled bars) strains after complementation with pPAPagL (pDEST JN105 derivative harboring P. aeruginosa pagL) is shown.

FIG. 4.

P. aeruginosa PagL enzymatic activity is induced in a clinical isolate by growth in medium limited for magnesium. Lipid A was isolated from a blood isolate of P. aeruginosa after growth in minimal medium supplemented with low (8 μM) or high (1 mM) magnesium concentrations and analyzed by MALDI-TOF MS in the negative-reflection ion mode. (A) Lipid A isolated after growth in high-magnesium medium resulted in the predominant ion species with a mass-to-charge ratio (m/z) of 1,419, consistent with a penta-acylated lipid A structure. (B) Lipid A isolated after growth in low-magnesium conditions gave a more complex spectrum consistent with the presence of both penta- (m/z = 1,447) and hexa-acylated ion species (m/z = 1,616, 1,685). *, location of the C12 fatty acid may be “piggyback” either at the 2- or 2′-position fatty acid on the P. aeruginosa lipid A structure. Adjacent peaks that differ by 16 m/z units represent the addition of an additional hydroxyl group (OH).

FIG. 5.

Deacylase enzymatic activity in laboratory-adapted P. aeruginosa strains is inhibited by growth at lower temperatures. (A) Lipid A was isolated from P. aeruginosa PAO-1 and PAK after growth in LB over a wide range of temperatures (15°C to 42°C), and total fatty acids were analyzed by gas chromatography. Levels of 3-OH C10 fatty acid are shown. (B) MS analysis of PAK lipid A isolated after growth at 15°C. (C) MS analysis of PAK lipid A isolated after growth at 42°C. (D) MS analysis of PAO-1 lipid A isolated after growth at 15°C. (E) MS analysis of PAO-1 lipid A isolated after growth at 42°C. Adjacent peaks that differ by 16 m/z units represent the addition of an additional hydroxyl group (OH).

FIG. 6.

Loss of deacylated P. aeruginosa lipid A species in some isolates from cystic fibrosis patients with severe pulmonary disease. Lipid A was isolated from clinical isolates of P. aeruginosa after growth in minimal medium supplemented with high (1 mM) magnesium concentrations and analyzed by MALDI-TOF MS in the negative-reflection ion mode. (A) MS analysis of lipid A isolated from clinical isolate CF565 after growth at 37°C; (B) MS analysis lipid A isolated from clinical isolate 10128 after growth at 37°C; and (C) MS analysis lipid A isolated from clinical isolate SE22 after growth at 37°C. Adjacent peaks that differ by 16 m/z units represent the addition of an additional hydroxyl group (OH).