Genetic Basis and Physiological Effects of Lipid A Hydroxylation in Pseudomonas aeruginosa PAO1.

Alessandra Lo Sciuto,Maria Concetta Spinnato,Alessandra Di Giamberardino,Matteo Cervoni,Francesco Imperi,Carmine Mancone,Roberta Stefanelli

doi:10.3390/pathogens8040291

Alessandra Lo Sciuto, Maria Concetta Spinnato + Show 5 more

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https://doi.org/10.3390/pathogens8040291

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Abstract

Modifications of the lipid A moiety of lipopolysaccharide influence the physicochemical properties of the outer membrane of Gram-negative bacteria. Some bacteria produce lipid A with a single hydroxylated secondary acyl chain. This hydroxylation is catalyzed by the dioxygenase LpxO, and is important for resistance to cationic antimicrobial peptides (e.g., polymyxins), survival in human blood, and pathogenicity in animal models. The lipid A of the human pathogen Pseudomonas aeruginosa can be hydroxylated in both secondary acyl chains, but the genetic basis and physiological role of these hydroxylations are still unknown. Through the generation of single and double deletion mutants in the lpxO1 and lpxO2 homologs of P. aeruginosa PAO1 and lipid A analysis by mass spectrometry, we demonstrate that both LpxO1 and LpxO2 are responsible for lipid A hydroxylation, likely acting on different secondary acyl chains. Lipid A hydroxylation does not appear to affect in vitro growth, cell wall stability, and resistance to human blood or antibiotics in P. aeruginosa. In contrast, it is required for infectivity in the Galleria mellonella infection model, without relevantly affecting in vivo persistence. Overall, these findings suggest a role for lipid A hydroxylation in P. aeruginosa virulence that could not be directly related to outer membrane integrity.

Highlights

The cell envelope of diderm (Gram-negative) bacteria consists of two concentric membranes, the cytoplasmic and the outer membrane, separated by the periplasmic space
While these lipid A forms were previously observed in P. aeruginosa [25,26,27], whether they have any effects on the properties of the LPS layer is not known
IPTG-induced expression of lpxO1 in the ∆lpxO1 mutant significantly increased the relative intensity of dihydroxylated lipid A peaks (Figure S1), suggesting that the minor effect of lpxO1 deletion on lipid A hydroxylation (Figure 2A) is likely due to poor expression and/or activity of the endogenous lpxO1 gene under the growth conditions tested in this work

Summary

Introduction

The cell envelope of diderm (Gram-negative) bacteria consists of two concentric membranes, the cytoplasmic (or inner) and the outer membrane, separated by the periplasmic space. The inner membrane is a typical phospholipids bilayer, while the outer membrane of most diderm bacteria is composed of glycerophospholipids and lipopolysaccharide (LPS) in the inner and outer leaflet, respectively [1]. Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen mostly dreaded for its high antibiotic resistance and for its ability to cause severe infections in immunocompromised patients. It represents the main cause of chronic lung infection in individuals suffering from cystic fibrosis, bronchiectasis, or chronic obstructive pulmonary disease [3]. P. aeruginosa LPS consists of a hydrophobic lipid A moiety, that anchors the molecule to the outer membrane, a conserved core oligosaccharide

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