Structural characterization of lipid A from nontypeable and type f Haemophilus influenzae: Variability of fatty acid substitution

Abstract

Lipid A isolated by mild acid hydrolysis from lipopolysaccharides of 22 nontypeable and 2 type f Haemophilus influenzae strains was investigated using electrospray ionization coupled to quadrupole ion trap mass spectrometry. The lengths, positions, and number of acyl chains in the lipid A molecule were determined using multiple-step tandem mass spectrometry (MS n ). All of the analyzed strains showed a major lipid A molecule comprising β-2-amino-2-deoxy- d-glucopyranose-(1 → 6)-α-2-amino-2-deoxy- d-glucopyranose phosphorylated at the C4′ and C1 positions. The C2/C2′ and C3/C3′ positions were substituted by amide-linked and ester-linked 3-hydroxytetradecanoic acid chains, respectively. The fatty acid chains on C3′ and C2′ were further esterified by tetradecanoic acid chains. In all strains, minor amounts of lipid A molecules with different acylation patterns were identified. Thus, structures comprising the hexaacylated lipid A with the C2 or C3 position being substituted by 3-hydroxydecanoic acid, and hexaacylated lipid A with the C3 and C3′ positions being substituted by 3-hydroxydodecanoic or dodecanoyloxytetradecanoic acid, respectively, were found. In addition, lipid A with an acetyl group attached to the 3-hydroxytetradecanoic acid groups attached to the C2 or C3 position was detected in two nontypeable H. influenzae strains.