Nature and location of amide‐bound (R)‐3‐acyloxyacyl groups in lipid A of lipopolysaccharides from various gram‐negative bacteria

Abstract

It has previously been demonstrated [Eur. J. Biochem. 124, 191-198 (1982) and 137, 15-22 (198311 that the lipid A component of Salmonella and Proteus lipopolysaccharides contains amide-linked (R)-3-acyloxyacyl residues. In the present study lipid A of other gram-negative bacteria was analysed for the presence of amidebound 3-acyloxyacyl residues. It was found that such residues are constituents of all lipid A tested (Agrobacterium tumefaciens, Chromobacterium violaceum, Pseudomonas aeruginosa, Xanthomonas sinensis, Bacteriodes frugilis, Vibrio cholerae, Fusobacterium nucleatum, Rhodospirillum tenue, Acinetobacter calcoaceticus, and Escherichia coli). Amide-linked (R)-3-acyloxyacyl groups, therefore, represent common and ubiquitous structural elements of bacterial lipid A.The composition of 3-acyloxyacyl groups differed considerably among different bacteria. As amide-bound (R)-3-hydroxy fatty acids straight chain and isobranched acyl groups with 10-17 carbon atoms were identified. The most frequently encountered fatty acids, substituting the 3-hydroxyl group of 3-hydroxy fatty acids, were nonhydroxylated straight chain and isobranched acyl residues with 10-17 carbon atoms as well as (Q-2-hydroxy fatty acids with 12 carbon atoms.In some cases, using laser desorption mass spectrometry, the distribution of 3-acyloxyacyl residues over the two available glucosamine amino groups of the lipid A backbone was investigated.Lipid A, the covalently bound lipid component of bacterial lipopolysaccharides (endotoxins) is, in general, made up by a bisphosphorylated p1 ,6-linked D-glucosamine disaccharide (lipid A backbone) which carries up to 4 mol of long-chain (R)-3-hydroxy fatty acid residues [I]. As was demonstrated for lipid A of Salmonella minnesota, Salmonella typhimurium, Escherichia coli and Proteus mirabilis 2 mol of these are linked to amino groups (positions 2 and 2') and 2 mol are bound to hydroxyl groups at positions 3 and 3' of the lipid A backbone 11-41. It has also previously been demonstrated that in these bacteria approximately 50% of the ester-linked (R)-3-hydroxy fatty acids are 3-0-acylated by straight-chain nonhydroxylated or (S)-Zhydroxy fatty acids [l, 3-61. Very recently it was shown for enterobacterial lipid A that also the amidebound (R)-3-hydroxy fatty acids (notably that at position 2') carry nonhydroxylated fatty acids at their 3-hydroxyl group in a specific distribution [7, 81. From these results it appeared that (R)-3-acyloxyacyl groups are important and dominating lipophilic constituents of lipid A and it was speculated that they may be also present in lipid A of bacteria other than Enterobacteriaceae.The studies reported in this paper, therefore, were performed to analyse lipid A of various bacterial origin for the presence and location of amide-linked (R)-3-acyloxyacyl residues. It will be shown that in all lipid A samples studied 3-acyloxyacyl groups are present indicating that they are common and prominent constituents of bacterial lipid A.Abbreviations. The abbreviations for fatty acids fo...

Biochemistry Of Endotoxins

“…1. The lipid A of enteric gram-negatives [E. co li (10--13), S. typ himurium (14,15), S. minneso ta (5 1, 64--66), and Proteus mira bilis (56,57,66)] is "asymmetrically" acylated in the sense that four fatty acids are associated with the nonreducing end, while only two are linked to the reducing end glucos amine ( Figure 3). With the exception of Hemophilus influenzae (67), the lipid A of the few non-enteric pathogens that have been characterized is "sym metrical" with respect to acyl chain placement ( Figure 3) (67)(68)(69)(70).…”

Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide

“…Quantitative analysis showed that, per mol GlcN, approximately 0.1 6 mol 3-(tetradecanoy1oxy)tetradecanoic acid and (Table 3). However, low yields of about 50% are associated with this method [44] and the reasons accounting for such yields have been discussed in detail elsewhere [51]. Since analysis by TLC showed that free dephosphorylated lipid A was heterogenous (composed of eight major species), therefore, prior to analysis by LDMS, it was subjected to silica-gel column chromatography.…”

Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid

“…Helander, Molecular Biology Unit, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland same holds true for studies of lipid A with laser desorption mass spectrometry [5,6]. To overcome these problems, chemical dephosphorylation of LPS by hydrofluoric acid is widely employed [4][5][6][7][8]. Typically a single treatment of LPS with aqueous HF brings about extensive dephosphorylation (>90°70), whereas the macromolecular structure of LPS is preserved indicating that the ester and amide linkages of fatty acids in lipid A and interglycosidic linkages in the polysaccharide chain are resistant to hydrofluoric acid.…”

“…Furthermore, phosphate groups can complicate the methylation analysis of LPS-derived oligosaccharides, and the Correspondence address: I.M. Helander, Molecular Biology Unit, National Public Health Institute, Mannerheimintie 166, SF-00300 Helsinki, Finland same holds true for studies of lipid A with laser desorption mass spectrometry [5,6]. To overcome these problems, chemical dephosphorylation of LPS by hydrofluoric acid is widely employed [4][5][6][7][8].…”