Characterization of a Novel Lipid-A fromRhizobium Species Sin-1

Abstract

The structure of the lipid-A from Rhizobium species Sin-1, a nitrogen-fixing Gram-negative bacterial symbiont of Sesbania, was determined by composition, nuclear magnetic resonance spectroscopic, and mass spectrometric analyses. The lipid-A preparation consisted of a mixture of structures due to differences in fatty acylation and in the glycosyl backbone. There were two different disaccharide backbones. One disaccharide consisted of a distal glucosaminosyl residue ␤-linked to position 6 of a proximal 2-aminoglucono-1,5-lactonosyl residue, and in the second disaccharide, the proximal residue was 2-amino-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactone. For both disaccharides, the distal glucosamine was acylated at C-2 primarily with ␤-hydroxypalmitate (␤-OHC16:0) which, in turn, was O-acylated with 27-hydroxyoctacosanoic acid. For some of the lipid-A molecules, the distal glucosaminosyl residue was also acylated at C-3 with ␤-hydroxymyristate (␤-OHC14: 0), whereas other molecules were devoid of this acyl substituent. Both the 2-aminoglucono-1,5-lactonosyl and 2-amino-2,3-dideoxy-D-erythro-hex-2-enono-1,5-lactonosyl residues were acylated at C-2, primarily with ␤-OHC16:0. Minor amounts of lipid-A molecules contained ␤-OHC14:0 at C-3 and/or ␤-hydroxystearate (␤-OHC18:0) or ␤-hydroxyoctadecenoate (␤-OHC18:1) as the C-2 and C-2 N-acyl substituents.Rhizobia refer collectively to the group of Gram-negative bacteria that belong to the Rhizobiaceae family and form nitrogen-fixing symbioses with legume plants. The major constituent of the Gram-negative bacterial cell wall is lipopolysaccharide (LPS).1 The LPS molecule has three structural regions as follows: the O-chain polysaccharide, core oligosaccharide, and the hydrophobic lipid-A. The LPS has been shown to be important in the symbiotic infection process (1-4). Structural changes to both the O-chain polysaccharide and to the lipid-A appear to be important for symbiotic infection (5). These changes include methylation of the O-chain glycosyl residues and increased fatty acylation of the lipid-A with 27-hydroxyoctacosanoic acid (27-OHC28:0) (6), a long-chain fatty acyl component that is common to the lipid-A isolated from members of the Rhizobiaceae (7-9). As with other Gram-negative bacteria, the LPS of rhizobia most likely have important roles that enable these bacteria to adapt to different environments; in this case, the intracellular environment of the legume host cell. These roles probably include acting as a permeation barrier toward potential toxins (e.g. defense response molecules from the host) as well as other structural adaptations that allow survival within the host cell. Lipid-A is considered the least variable region in the LPS molecule. The lipid-A structure from enteric bacteria is largely conserved, consisting of a ␤-(136)-linked glucosamine disaccharide backbone with phosphate groups at C-1 and C-4Ј and ␤-hydroxy fatty acyl groups and acyloxyacyl residues at positions 2 and 3, and 2Ј and 3Ј, respectively (10, 11). Modifications to this structure that are...

Rhizobium Sin-1 Lipopolysaccharide (LPS) Prevents Enteric LPS-induced Cytokine Production

“…The Rhizobium Sin-1 LPS preparation used contains a variety of lipid-A moieties with minor structural differences. The mixture of possible lipid-A structures in Rhizobium Sin-1 LPS are described in the companion paper (39) and shown in Fig. 6B.…”

“…Fractions containing purified rhizobial LPS were pooled, dialyzed against water, and stored lyophilized. The purity of the LPS was determined by deoxycholate-PAGE analysis and silver staining, as well as by glycosyl and fatty acyl residue analysis as described previously (39). Cell Culture Techniques-Mono Mac 6 cells, a human monocytic cell line, were kindly provided by Dr. H. W. L. Ziegler-Heitbrock (University of Munich, Germany) (26,27).…”

“…For both R. etli CE3 and Rhizobium Sin-1, the structures shown are those that are likely to be present in the intact LPS. Additional structures are present in lipid-A isolated by mild acid hydrolysis of the LPS that may be a result of the hydrolysis procedure (37)(38)(39). There are also variations on these structures due to the fact that the N-acyl substituents can be 3-OHC14:0, 3-OHC16:0, or 3-OHC18:0 (A) and that the proximal 2-aminogluconate residue may also be present as a 2-amino-1,5-gluconolactone (B).…”

“…Having identified a structurally novel LPS from nitrogen-fixing bacteria, we were interested in determining the effects of these novel rhizobial LPS molecules on human monocytic cells, and whether individual rhizobial LPS might antagonize enteric LPS-induced synthesis of TNF␣ by these cells (23,24). In particular, the lipid-A of Rhizobium Sin-1 differs significantly from other well characterized lipid-A analogs in its unique carbohydrate backbone, fatty acid acylation patterns, and the lack of phosphate (39). These structural differences define Rhizobium Sin-1 lipid-A as a potentially novel LPS antagonist that might serve as a therapeutic agent for the prevention of circulatory shock due to Gram-negative sepsis.…”

Agonistic and antagonistic properties of a Rhizobium sin-1 lipid A modified by an ether-linked lipid

“…1). 21 In particular, the disaccharide moiety of Rhizobial lipid A is devoid of phosphate and the glucosamine phosphate is replaced by 2-aminogluconolactone. It contains a very long chain fatty acid, 27-hydroxyoctacosanoic acid which, in turn, can be esterified by β-hydroxybutyrate.…”