The chemical structure of Bacteroides fragilis NCTC 9343 lipid A was characterized by using conventional chemical procedures, methylation analysis, and laser desorption mass spectrometry. It was found that B. fragilis lipid A consists of a P-D-glucosaminyl-(l-6)-~-glucosaminyl-l-O-phosphate backbone whose hydroxyl groups in positions 4, 4' and 6' are free, the latter serving as the attachment site for the polysaccharide component in lipopolysaccharide. This backbone molecule carries up to of five molecules of ester-and amide-bound long chain non-hydroxylated and (R)-3-hydroxy fatty acids. With regard to the distribution of the fatty acids on the lipid A backbone, a considerable heterogeneity was revealed by laser desorption mass spectrometry. Despite this heterogeneity, a major species of B. fragilis lipid A could be defined in which the hydroxyl group at position 3' of the distal GlcN carries (R)-3-hydroxyhexadecanoic acid and the hydroxyl group at position 3 of the reducing GlcN is acylated by (R)-3-hydroxypentadecanoic acid. The amino group of the distal GlcN residue carries (R)-3-( 13-methyltetradecanoyloxy)-1.5-methylhexadecanoic acid and that of the reducing GlcN group (R)-3-hydroxyhexadecanoic acid. The absence of ester-bound phosphate and ester-linked 3-acyloxyacyl groups, the presence of not more than five acyl residues and the predominance of fatty acids possessing 15 -17 carbon atoms are unique features of B.,fragilis lipid A which differentiate it from enterobacterial and other lipids A and which are likely to be related to its low endotoxic activity.Bacteroides fragilis is the predominant bacterial isolate in anaerobic septicaemic states [l]. In the mediation of the effects of Gram-negative septicaemia, endotoxins are likely to play a major role [2]. The endotoxin of B. fragilis has previously been studied and shown to be chemically a lipopolysaccharide consisting of a short polysaccharide chain and a lipid A component [3, 41. The endotoxic potency of B. fragilis lipopolysaccharide, however, was found to be low as compared, for example, to enterobacterial preparations [5 -71.It is now well established that the endotoxic properties of lipopolysaccharides are mediated by their lipid A component and, further, that endotoxic activity depends on a defined structural make up of lipid A [8, 91. In view of the low endotoxic activity of B. , fragilis lipopolysaccharide we reasoned that the structure of its lipid A component may differ from that of active lipids A like that of Escherichia coli [8, 91.The present study was performed in order to characterize the chemical structure of B. fragilis lipid A. It will be shown that indeed B. fragilis lipid A exhibits a quite different phosphorylation and acylation pattern, as compared to enterobacterial lipid A. The abbreviations for fatty acids follow IUPAC recommendations [see Eur. J . Biochem. 79, 20 (1977)]. MATERIALS AND METHODS Bacteria and lipopolysaccharideBacteroides fragilis strain NCTC 9343 was obtained from the National Collection of Type Cultures (London...

No abstract

In Salmonella minnesota lipopolysaccharide the lipid A backbone, a substituted diphosphorylated p1 ,6-linked D-glucosamine disaccharide molecule, carries approximately seven residues of fatty acids : one each of dodecanoic, hexadecanoic, D-3-hydroxytetradecanoic and ~-3-O-(tetradecanoyl)-tetradecanoic acid in ester linkage and two of D-3-hydroxytetradecanoic acid in amide linkage. In the present study it is shown that treatment of the lipopolysaccharide with alkali at elevated temperature leads, through a p-elimination reaction, to the generation of amidebound A'-tetradecenoic acid. This suggested that the 3-hydroxyl group of amide-bound hydroxy fatty acids carried a substituent. To elucidate the nature of the substituent, free Salmonella lipid A was methylated with methyl iodine in the presence of silver salts followed by mild acid hydrolysis, a procedure which is known to cleave amide (and not ester) bonds selectively. In the hydrolysate, by means of combined gas-liquid chromatography/mass spectrometry the methyl esters of 3-O-(dodecanoyl)-tetradecanoic and 3-O-(hexadecanoyl)-tetradecanoic acid were identified. This shows that in lipid A amide-linked 3-hydroxytetradecanoic acid residues are 3-0-acylated by dodecanoic and hexadecanoic acid, respectively.Quantitative analyses suggest that the Salmonella lipid A backbone is substituted by four D-3-hydroxytetradecanoyl residues, two being present as esters and two as amides. The nonhydroxylated fatty acids are not bound directly to the backbone. Rather, they are attached to hydroxyl groups of 3-hydroxytet radecanoyl residues : specifically, tetradecanoic acid substitutes ester-bound and dodecanoic and hexadecanoic acid amide-bound 3-hydroxytetradecanoic acid.Lipopolysaccharides, the 0-antigens and endotoxins of gram-negative bacteria, are made up of two chemically distinct portions : a heteropolysaccharide and a covalently linked lipid, termed lipid A [l]. While the structures determining 0-antigenicity are embedded in the polysaccharide part, the endotoxic properties of lipopolysaccharides are anchored in the lipid A component [2]. Chemically, lipid A of Salmonella (and many other gram-negative bacteria) consists of a p1 ,6-linked D-glucosamine disaccharide which carries partially substituted phosphoryl ksidues in positions C-4' and C-1. The hydroxyl group at C-3' represents the attachment site of the saccharide portion in intact lipopolysaccharide. The phosphorylated glucosamine disaccharide (lipid A backbone) carries, in addition, long-chain fatty acids in both ester and amide linkage which appear to play a role in the expression of endotoxic activity. Of the acyl residues present in Salmonella lipid A approximately one each of dodecanoic, hexadecanoic, D-3-hydroxytetradecanoic and ~-3-O-(tetradecanoyI)-tetradecanoic acid (and possibly smaller amounts of ~-3 -0 -(~-2 -hydroxytetradecanoy1)-tetradecanoic acid) are involved in ester and two of D-3-hydroxytetradecanoic acid in amide linkage [3].According to our present knowledge, the Salmonella lipid A backbone pr...

No abstract

In the present paper laser desorption mass spectrometry (LDMS) was applied to dephosphorylated free lipid A preparations obtained from lipopolysaccharides of Re mutants of Salmonella minnesota, Eschel-icliici coli and Proteus mirahilis. The purpose of this study was to elucidate the location of (R)-3-hydroxytetradecanoic acid and 3-0-acylated (R)-3-hydroxytetradecanoic acid residues which are bound to amino and hydroxyl groups of the glucosamine disaccharide backbone of lipid A. Based on the previous finding from biochemical analyses that the amino group of the nonreducing glucosamine residue (GlcN 11) of the backbone carries, in S. minnesotu and E. coli, 3-dodecanoyloxytetradecanoic acid and, in P. mirabilis, 3-tetradecanoyloxytetradecanoic acid, a self-consistent interpretation of the LDMS was possible.It was found that: (a) in all three lipids A GlcN I1 is, besides the amide-linked 3-acyloxyacyl residue, substituted by ester-linked 3-tetradecanoyloxytetradecanoic acid; (b) the reducing glucosamine (GlcN I) is substituted by ester-linked 3-hydroxytetradecanoic acid; (c) the amino group of GlcN I carries a 3-hydroxytetradecanoic acid which is non-acylated in E. coli and which is partially acylated by hexadecanoic acid in S. rninnesota and P. mirabilis.In lipids A which were obtained from the P. mirahi1i.s Re mutant grown at low temperature (12 C) LDMS analysis revealed that specifically the one fatty acid bound to the 3-hydroxyl group of amide-linked 3-hydroxytetradecanoic acid at GlcN I1 is positionally replaced by d9-hexadecenoic acid (palmitoleic acid).It appears, therefore, that enterobacterial lipids A resemble each other in that the 3-hydroxyl groups of the two 3-hydroxytetradecanoic acid residues linked to GlcN I1 are fully acylated, while those of the two 3-hydroxytetradecanoic acid groups attached to GlcN I are free or only partially substituted.