A Novel Secondary Acyl Chain in the Lipopolysaccharide of Bordetella pertussis Required for Efficient Infection of Human Macrophages

Abstract

Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2 and 3 positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2 position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.Pertussis or whooping cough is a severe acute respiratory illness that is characterized by paroxysmal coughing and a distinctive "whooping" sound when air is subsequently inhaled. The disease is highly contagious and most severe in neonates and children younger than one year. Pertussis is caused by the Gram-negative bacterium Bordetella pertussis. While the genus Bordetella currently encompasses nine species, apart from B. pertussis only three other members, Bordetella bronchiseptica, Bordetella parapertussis, and Bordetella holmesii, have been associated with respiratory infections in humans and other mammals (1). The Gram-negative bacterial cell envelope is composed of two membranes, the inner and the outer membrane, which are separated by the periplasm. The inner membrane is a symmetrical bilayer composed of phospholipids, whereas the outer membrane is asymmetric and consists of phospholipids in the inner leaflet and lipopolysaccharide (LPS) 2 in the outer leaflet. LPS, which is also known as endotoxin, consists of three distinct structural domains: lipid A, the core, and the O-antigen (2). The first domain, lipid A, functions as a hydrophobic membrane anchor and forms the bioactive component of the molecule (3). The structure of lipid A is conserved among different bacterial groups, indicating its importance for the correct functioning of the outer membrane. Generally, lipid A consists of a ␤-1Ј,6-linked D-glucosamine (GlcN) disaccharide carrying ester-and amide-linked 3-hydroxyl fatty acids at the C-2, C-3, C-2Ј, and C-3Ј positions, and phosphate groups at positions C-1 and C-4Ј. The endotoxic activity of LPS is based on the recognition of lipid A by the TLR4/MD-2 complex of the host, which leads to the activation of NF-B and, consequently, to an increased production and secretion of proinflammatory cytokines, such as IL-6, tumor necrosis factor-␣, and IL-1␤ (4). C...

Structural characterization of Bordetella parapertussis lipid A

“…Recently, the occurrence of a novel secondary acyl chain in B. pertussis LPS and its negative effect on infection of human macrophages has been reported (19).…”

“…The lipid A molecules can be modified via acylation; deacylation; secondary fatty acid hydroxylation; and /or phosphate-group substitutions with aminoarabinose, galactosamine, or phosphoethanolamine (17)(18)(19). We recently reported substitution with glucosamine on the phosphates of B. bronchiseptica and B. pertussis lipids A, causing on other examples a significant modulation of host responses to infection (11,20).…”

Deciphering tissue-induced Klebsiella pneumoniae lipid A structure

“…The presence of 2-hydroxyl fatty acids in the lipid A has been reported for Salmonella enterica serovar typhimurium (C 14:OH ) (5,7,8), Pseudomonas aeruginosa (C 12:OH ) (17), and Bordetella spp. (C 12:OH ) (18,19). LpxO has been identified as the oxygenase that 2-hydroxylates the C 14 or C 12 acyl chains.…”

Bordetella pertussis Naturally Occurring Isolates with Altered Lipooligosaccharide Structure Fail To Fully Mature Human Dendritic Cells

“…After freeze-drying, the pellets were washed twice with methanol and centrifuged (4,600 ϫ g for 20 min). The pellets were extracted using chloroform-methanol-water (12:6:1, vol/vol/vol), centrifuged (4,600 ϫ g for 20 min), and the lipid A extract in the upper phase was analyzed by electrospray ionization mass spectrometry (ESI-MS) with a Finnigan LCQ instrument in the negative-ion mode (39).…”