Structure of Cell Wall Lipopolysaccharide from Salmonella typhimurium. Further Studies on the Linkage between O Side Chains and R Core

Abstract

Cell wall lipopolysaccharide from a wild type Salmonella typhimurium and its Ra mutant were methylated and hydrolyzed, and methylated sugars were examined by gas-liquid chromatography after reduction and acetylation. A peak from the wild type lipopolysaccharide, previously believed to be the derivative of 3,6-di-O-methyl-~-galactose, was found to be derived from 3,6-di-O-methyl-~-glucose by the use of uniformly 14C-labeled lipopolysaccharide and of lipopolysaccharide specifically labeled with 14C in its galactose residues. Ra mutant lipopolysaccharide, which lacks the 0 side chain portion, did not produce the above-mentioned derivative but produced upon methylation a sugar, tentatively identified from its retention time as 3,4,6-tri-O-methyl-D-glucose. These results are consistent with our earlier proposal that 0 side chains are linked to C-4 of the subterminal D-glucose residue of R core portion of lipopolysaccharide, and further indicate that this glucose residue is also substituted a t C-2, presumably by the non-reducing terminal N-acetyl-D-glucosamine residue of the R core.Cell wall lipopolysaccharide from Salmonella consists of three regions [l] (Fig. 1). The outermost region, 0 side chain, which is composed of oligosaccharide repeat units, is linked to the R core polysaccharide, which is then linked to a lipid, called lipid A. I n a preceding paper of this series, it was shown, by periodate oxidation, that 0 side chains are However, owing to the limitations inherent in the methods used, the conclusion was not definite and confirmation by other methods was thought desirable. This paper describes the results obtained by one such method, i.e. methylation analysis.If the structure of linkage region proposed earlier is correct (see Fig. l), we can make several predictions on the outcome of methylation of cell wall lipopolysaccharide followed by acid hydrolysis. The methylation analysis of complex heteropolysaccharides was greatly improved by Lindberg and his coworkers, who converted methylated sugars into 0-acetyl-0-methyl-alditois before gas-liquid chromatography, and identified peaks by mass spectrometric analysis. A thorough analysis of Salmonella typhimurium cell wall lipopolysaccharide by this technique has been published [3,4], but none of the expected derivatives of subterminal glucose was seen. No tri-0-methyl-glucose was found, and although one di-0-methyl-glucose was seen, it had 0-methyl groups on C-2 and (2-4, whereas the expected di-0-methyl-glucose from the linkage region should not be methylated a t C-4 as this position would be blocked by the 0 side chain [2]. METHODS Bacterial StrainsSalmonella typhirnurium LT2 and its derivatives were nsed. TV205 is a rfb mutant, synthesizing an Ra-type lipopolysaccharide composed of a complete

Biosynthesis of Cell Wall Lipopolysaccharide in Mutants of Salmonella V. A Mutant of Salmonella typhimurium Defective in the Synthesis of Cytidine Diphosphoabequose

“…It is possible that genes for mannosyl transferase and abequosyl transferase are also located in this region. Since the polar mutation rfbH8J9 lowers, by about 30%, the activity of an enzyme (MAN-3; Table 4) coded for by the gene rfbM located to the right of rfbH (17), the mutation should also lower the (15) a Only dTDP-rhamnose and UDP-galactose were added for the assay of rhamnose incorporation; only GDP-mannose, dTDP-rhamnose, and UDP-galactose were present in the assay mixture for mannose incorporation. Abequose incorporation was assayed under a quite different set of conditions, and the absolute amounts of abequose incorporation cannot be compared with the amounts of mannose or rhamnose incorporation.…”

“…This scheme, which shows the structure of LPS from S. typhimurium containing 0 antigens 4 and 12 but lacking 0 antigens 1, 5, and 122, is based on the results from several laboratories (for review, see reference 11). The 0 side chain was recently found to be linked to the subterminal glucose residue of the R core (15). Abbreviations: Abe, abequose; Rha, L-rhamnose; Gal, D-galactose; Man, Dmannose; Glc, glucose; GkcNAc, N-acetyl-D-glucosamine: Hep, L-glycero-D-mannoheptose: KDO, 3-deoxyoctulosonate: P, phosphate.…”

Enhancement of Mite Allergen-Induced Eosinophil Infiltration in the Murine Airway and Local Cytokine/Chemokine Expression by Asian Sand Dust

“…On the other hand, LPS and β-glucan were adsorbed on the sand dust particles. LPS is a glycolipid of gram-negative bacteria (Nikaido, 1969), and β-glucan is the major structural component of fungi walls (Hearn & Sietsma, 1994). These microbial materials induced neutrophilic pulmonary inflammation (Takano et al, 2002;Young et al, 2001).…”

Experimental Salmonellosis: Hypersensitivity to Cell Wall Lipopolysaccharide and Anti-Infectious Resistance of Mice Infected with Salmonella

“…Each group of 10 mice was kept in a cage containing sterilized wood shavings and was given autoclaved mouse diet (MF, Oriental Yeast Co., Tokyo, Japan)j and drug-free water. Natural infection with Salmonella was checked by cul- (13). The virulence and immunizing potencies of these bacteria have been reported elsewhere (6).…”