Investigation of the structure of lipid A from Actinobacillus actinomycetemcomitans strain Y4 and human clinical isolate PO 1021‐7

Abstract

The lipopolysaccharides of Actinobacillus actinomycetemcomitans strain Y4 and a human clinical isolate PO 1021‐7 were examined by SDS/PAGE, deoxycholate/PAGE and mass spectrometry. PAGE analysis revealed an electrophoretic pattern similar to the SR‐type lipopolysaccharide (LPS) of Salmonella. Deoxycholate/PAGE indicated the LPS of A. actinomycetemcomitans to consist of short sugar chains. Chemical analysis revealed the presence of thiobarbituric‐acid‐positive material (3‐deoxy‐d‐manno‐octulosonic acid equivalents) and four neutral sugars: glucose, galactose, d‐glycero‐d‐manno‐heptose and l‐glycero‐d‐manno‐heptose. Phosphate, glucosamine, glycine, and the fatty acids, 3‐hydroxymyristic acid, myristic acid and palmitic acid, comprised the remainder of the molecule. The structure of the free lipid A revealed it to consist of a 1,6‐glucosamine disaccharide esterified at C4′ by a phosphomonoester. The hydroxyl group at C3 and the amide group of the non‐reducing glucosamine were both acylated by 3‐myristoylmyristic acid; analogous sites on the reducing glucosamine were acylated by 3‐hydroxymyristic acid. Hydroxyl groups at C4 and C6′ in the free lipid A were unsubstituted, with C6 being the proposed attachment site of the polysaccharide moiety. Chemical analysis revealed the presence of glycine in the intact LPS; its exact location in the A. actinomycetemcomitans LPS is still to be determined. Both intact LPS and free lipid A were highly lethal to galactosamine‐sensitized mice, comparable to that of Salmonella.