Site-specific activity of the acyltransferases HtrB1 and HtrB2 in Pseudomonas aeruginosa lipid A biosynthesis

Abstract

Pseudomonas aeruginosa (PA) is an opportunistic Gram-negative pathogen associated with nosocomial infections, acute infections and chronic lung infections in patients with cystic fibrosis. The ability of PA to cause infection can be attributed to its ability to adapt to a multitude of environments. Modification of the lipid A portion of lipopolysaccharide (LPS) is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli. Lipid A, the endotoxic moiety of LPS, is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria making it a critical factor for bacterial adaptation. One way PA modifies its lipid A is through the addition of laurate and 2-hydroxylaurate. This secondary or late acylation is carried out by the acyltransferase, HtrB (LpxL). Analysis of the PA genome revealed the presence of two htrB homologs, PA0011 (htrB1) and PA3242 (htrB2). In this study, we were able to show that each gene identified is responsible for site-specific modification of lipid A. Additionally, deletions of either gene altered resistance to specific classes of antibiotics, cationic antimicrobial peptides and increased membrane permeability suggesting a role for these enzymes in maintaining optimal membrane organization and integrity.

Keywords: HtrB; LPS; Pseudomonas aeruginosa; acyltransferase; lipid A; membrane remodeling.

Graphical Abstract Figure.

Modification of the lipid A portion of lipopolysaccharide through altered acylation is a vital mechanism Gram-negative pathogens use to remodel the outer membrane in response to environmental stimuli.

Figure 1.

Synthesis of PA lipid A. Initial modifications to the tetraacylated lipid A molecule include the addition of laurate and 2-hydroxylaurate. Removal of the 3 position 3-hydroxydecanoate results in the highest abundance PA lipid A species.

Figure 2.

MALDI-TOF MS of (A) WT major ion peak m/z 1446.8; (B) ΔhtrB1 major peak m/z 1248 corresponding to a loss of 2-hydroxylaurate at the C-2 position; (C) ΔhtrB2 major peak at m/z 1264 resulting from a loss of laurate at the C-2’ position.

Figure 3.

GC analysis of lipid A isolated from WT (white bars), ΔhtrB1 (gray bars) and ΔhtrB2 (black bars). A loss of C12 was seen in ΔhtrB2 while the levels of 2OH-C12 were diminished in the ΔhtrB1 mutant.

Figure 4.

Growth curves of (A) WT (solid line), (B) ΔhtrB1 (dashed line), and (C) ΔhtrB2 (dotted line) at 25, 37 and 42°C. Curves are shown as the average of three biological replicates.

Figure 5.

Ethidium bromide uptake assay measuring the permeability of ΔhtrB1 (gray bars) and ΔhtrB2 (black bars) cell membranes. Passive permeability with active efflux pump activity was measured by EtBr alone. EtBr plus the efflux pump inhibitor CZP measured passive permeability alone.

Figure 6.

Inhibition of WT, ΔhtrB1 and ΔhtrB2 by polymyxin B (black bars) and colistin (gray bars). Each experiment was carried out in triplicate.

Figure 7.

Antibiotic susceptibility of WT (white bars), ΔhtrB1 (gray bars) and ΔhtrB2 (black bars) measured by an MIC assay in Mueller Hinton broth. Antibiotics tested included cell wall synthesis inhibitors (carbenicillin, ticarcillin, piperacillin and cefoperazone), protein synthesis inhibitors (chloramphenicol and minocycline), RNA polymerase inhibitor (Rifampicin) and DNA gyrase inhibitor (ciprofloxacin).