Structural Investigations on the Core Polysaccharide of Salmonella typhimurium and the Mode of Attachment of the O-Specific Chains

Abstract

Two lipopolysaccharides were analyzed comparatively : the lipopolysaccharide derived from a Salmonella typhimurium SR mutant, containing single repeating units linked to the core polysaccharide, and a lipopolysaccharide derived from a corresponding Ra mutant containing core polysaccharide only. Partial hydrolysis of these lipopolysaccharides led to the formation of a group of oligosaccharides occurring in both lipopolysaccharides. Their structural analysis provided information for the reconstruction of the hexose region of the core. From the SR lipopolysaccharide, four oligosaccharides were identified which were absent from the Ra lipopolysaccharide: (a) Man-al,4-rhamnose (derived from the repeating units) ; (b) Gal-j31,4-Glc ; (c) Gal-B1,4-(GlcNAc-a1,Z-)Glc ; (d) Gal-f?l,4-Glc-ccl,2-Gal. Oligosaccharides (b), (c), and (d) are derived from the linkage region between the repeating unit and the core. Methylation studies which were performed on the SR and Ra lipopolysaccharides led to the identification of the sugar linkages. On the basis of the present investigation, the partial structure of the SR lipopolysaccharide can be drawn as shown below, which confirms and extends previous results. The findings are discussed in regard to the biosynthesis and immunochemistry of SR and S form lipopolysaccharides. Abe-OAcGlcNAc (Glc) GalMan-ccl,4-Rha-~l,3-Gal-~l,4-Glc-al,2-Gal-ccl,3-Glc ------ Fig. 1). Glucose 11, therefore, represents a branching point ; it is substituted a t positions 2 and 4 [2], carrying N-acetylglucosamine as a small side chain (Fig. 1). The work described in this paper was performed with the aim of obtaining more information on the structure of Salmonella lipopolysaccharides, and, especially, on the linkage between 0-specific chains and the core. For this purpose, the lipopolysaccharides of two S. typhimurium mutants were compared :(a) The Ra mutant his 386, which is a deletion mutant defective in the rfb locus and which, therefore, produces a lipopolysaccharide containing, besides lipid A, only the complete core polysaccharide [3]. (b) The SR mutant SH777, which is defective in the rfc locus, and which produces a lipopolysaccharide containing the complete core, to which is attached one single 0-specific repeating unit (in contrast to the wild form lipopolysaccharide, which contains a sequence of repeating units linked to the core) [4].The analysis of oligosacchafide fragments, obtained by partial hydrolysis of the two lipopolysaccharides, as well as the results of methylation studies, confirmed the previously proposed structures of the core [5] and of the linkage region between the repeating unit and the core [2]. Furthermore, the results led to certain new details regarding sugar linkages in these lipopolysaccharides. It was found that in the SR lipopolysaccharide, the glucose I1 residue carries the N-acetylglucosamine residue a t position 2, and the terminal reducing galactose residue of the repeating unit a t position 4. Further-

Bacteriophage-mediated Glucosylation Can Modify Lipopolysaccharide O-Antigens Synthesized by an ATP-binding Cassette (ABC) Transporter-dependent Assembly Mechanism

“…Biosynthetic experiments suggested that the acceptor for the modification reaction is the Und-PP-linked O-antigen polymerization product rather than the individual Und-PP-linked repeat units (12). Consistent with this proposal, the LPS products of wzy mutants, which contain a single O-repeat unit, are not modified in Salmonella (50,51). Likewise, the first repeat unit of S. flexneri O-antigen is not subject to glucosylation (7,53,54).…”

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

“…On the average SH2183 LPS contained six repeats of this oligosaccharide, since approximately six D-Man and L-Rha residues were found per mol of LPS. The lower proportion of Abe is attributable to its known instablity in neutral sugar analysis [22], whereas D-GIc and D-Gal are constituents of the core oligosaccharide as well. The reducing end of lipid A was quantitatively substituted, as indicated by the lack of reactivity in the direct Morgan-Elson assay.…”

“…The hydrofluoric acid treatment probably cleaved also a significant portion of the branch sugar (Abe, also a deoxysugar). The glycosidic linkages provided by L-Rha in the O antigen are known to be susceptible to hydrolysis by 0.5 M H2SO4 at 100°C so that oligosaccharides with terminal L-Rha are produced; such treatments, however, also produce oligosaccharides stemming from the core [26,27]. It is apparent from the results that the ketosidic linkage provided by the deoxysugar dOclA, which is known to be highly labile towards acid hydrolysis by HCI or CH3COOH [1,2], was resistant to hydrofluoric acid.…”

Studies on the Structure of T1 Lipopolysaccharides

“…The results of methylation analysis performed on this fragment showed that the ribose chains are linked either directly or indirectly to the 4-position of the subterminal sugar of the core, i.e. glucose 11, which had also been identified previously [3,20] as the attachment site of Ti-specific chains. It cannot yet be decided, however, whether the ribose chain is linked directly to the core, or whether this linkage is formed via one or a few galactose units.…”