Structural Elucidation of the Biological Repeating Unit of O-specific Polysaccharide from Citrobacter Serotype O41

Abstract

Sugar analysis of the 0-specific polysaccharide produced by Citrobacter serotype 0 4 1 revealed the presence of a hexasaccharide repeating unit, which includes the unusual 3-amino-N-(~-3'-hydroxybutyryl)-3,6-dideoxy-~-galactosyl residue (Fuc3NAcyl). The structure of the repeating unit was determined by extensive use of homo-and heteronuclear two-dimensional NMR spectroscopy, including the application of long-range 'H-I3C correlation experiments and NOE studies to establish the sequence of sugar units. Identification of the Glc~l--.2Fuc3NAcyl~1--.6GlcNAcal~ sequence at the non-reducing terminus establishes the biological repeating unit of this 0-specific polysaccharide as : +2Glc~l+2Fuc3NAcyl-~1+6GlcNAcal-(Glcul+2)4Gal~l--.3GalNAc~l+. This structure is similar to that found for the 0-specific polysaccharide isolated from Hafnia alvei strain 121 1 [Katzenellenbogen, E., Romanowska, E.,Biochem. 200,401 -4071, which differs in having an acetyl substituent at 04 of the Fuc3NAcyl residue and a branch point of GalNAca substituted by GlcP at 0 3 ; these differences are responsible for the only weak serological cross-reactivity of the two strains.Keywords. Lipopolysaccharide; endotoxin; Citrobacter; 3-amino-N-(~-3'-hydroxybutyryl)-3,6-dideoxy-This work is part of a research program investigating the endotoxins produced by serotypes of the Gram-negative bacterium Citrobacter (Romanowska et al., 1990). Although these bacteria appear to be normal inhabitants of the intestinal tract of man and animals, they are associated with infections of the gut, urinary tract, gall bladder, middle ear and meninges (Sedlak, 1974;Romanowska et al., 1990). The genus Citrobacter has been divided into 42 serotypes, and their lipopolysaccharides into 20 chemotypes, on the basis of serological and immunological studies. As part of our systematic study to explain the serological diversity observed and thereby establish structure/activity relationships, we now present the structural elucidation of the 0-specific polysaccharide isolated from Citrobacter serotype 041. This strain belongs to the CC-D chemotype, and is characterized by the presence of 3-amino-3,6-dideoxy-galactose (Keleti et al., 1971). In view of the complexity of the structure to be determined and to explore the advantages of different assignment strategies, we complemented our usual approach to the delineation of endotoxin structures using 'H-NMR methods by extensive use of heteronuclear NMR experiments, including direct and long-range 'H-13C correlation measurements.