O-specific polysaccharide was isolated from Proteus penneri strain 12 (ATCC 33519) lipopolysaccharide (LPS) and studied using NMR spectroscopy, including selective spin-decoupling, one-dimensional NOE, two-dimensional homonuclear correlation spectroscopy, 13C,1H heteronuclear correlation spectroscopy and chemical methods (O-deacetylation, Smith degradation, partial acid hydrolysis followed by borohydride reduction and methylation). The amide of D-galacturonic acid with L-threonine [D-GalA(L-Thr)] was identified as a constituent of the polysaccharide and the following structure of the tetrasaccharide repeating unit was established: [formula: see text] where the degree of O-acetylation at either position varies over 20-40%. Serological study with LPS, its degradation products and related synthetic glycoconjugates (2-acrylamidoethyl glycosides of amides of alpha-D-GalA with L-amino acids copolymerised with acrylamide) showed that D-GalA(L-Thr) plays an important role in manifesting the serological specificity of the P. penneri 12 O-antigen. Serological cross reactions between LPSs of P. penneri 12 and Proteus mirabilis S1959, R14/S1959 (transient-like form), O23 and O28 are discussed.
The 0-specific polysaccharide of Proteus mirabilis 0 2 8 was found to contain D-galactose, D-gdaCtUronic acid (GalA j, 2-acetamido-2-deoxy-~-glucose, L-serine, L-lysine, and 0-acetyl groups in molar ratios 1 : 2 : 1 : 1 : 1 : 1, the amino acids being linked via their a-amino group to the carboxyl group of GalA. The polysaccharide was studied using 'Hand 13C-NMR spectroscopy, including selective spin-decoupling, one-dimensional total correlation spectroscopy, two-dimensional homonuclear correlation spectroscopy (COSY), heteronuclear I3C,'H COSY, one-dimensional NOE, and two-dimensional rotating-frame NOE spectroscopy and partial acid hydrolysis followed by borohydride reduction, methylation, and GLCMS analysis of the derived glycosyl alditols. The following structure of the repeating unit was established: L-LYS L-Ser 6 6 4 OAc I I -4)-a-~-GalpA-( 1-4)-a-~-Galp-( 1-.3)-a-~-GalpA-( 1+3)-P-~-GlcpNAc-( 1-
IEpitope specificity of the I? mirabilis 0 2 8 polysaccharide was analysed using a homologous rabbit polyclonal antiserum in quantitative precipitation, passive immunohemolysis, and inhibition of passive immunohemolysis. Study with related synthetic glycopolymers (2-acrylamidoethyl glycosides of amides of a-D-GalA with amino acids copolymerised with acrylamidej showed the importance of D-G~A(L-LYS) for manifesting serological specificity of the 0-antigen. Serological cross-reactions between P. mirabilis 028, S1959, and R14/S1959 (a transient-like form) are discussed.
@Specific polysaccharide was obtained by mild acid degradation of Proteus penneri strain 16 lipopolysaccharide and found to contain D-glucose, D-glucuronic acid, 2-acetam~do-2-deoxy-~-glucose, and 3,6-dideoxy-3-[(R)-3-hydroxybutyramido]-~-galactose in the ratio of 2: 1 : 1 : 1 as well as a small proportion of 0-acetyl groups. On the basis of one-dimensional 'H-NMRI3C-NMR and NOE spectroscopy, two-dimensional homonuclear-shift-correlated spectroscopy with one-step and two-step relayed coherence transfer and heteronuclear 'H/' 3C NMR shift-correlated spectroscopy, it was concluded that the 0-specific polysaccharide of P. penneri strain 16 has the following structure:This structure was confirmed by methylation analysis and structural analysis of a linear tetrasaccharide fragment prepared by cleavage of the polysaccharide with anhydrous hydrogen fluoride followed by conversion of the a-tetrosyl fluoride obtained in to the corresponding free oligosaccharide and alditol. 0-Acetyl groups were tentatively located at position 3 of the glucuronic acid residue and at position 4 of the 6-substituted glucose residue, the degree of acetylation being less than 20% of the total.Cross-reactions of P. penneri strain 16 anti-(0-specific polysaccharide) antiserum with lipopolysaccharides from several other Proteus strains and the role of 3,6-dideoxy-3-(R)-3-hydroxybutyramido-~-galactose in the serological specificity of P. penneri strain 16 are discussed.Recently the name Proteus penneri has been proposed for strains formerly called Proteus vulgaris biogroup 1 [l, 21. The, genetic and metabolic differences of the novel Proteus species in comparison with other species of this genus could be reflected in the composition and structures of their lipopolysaccharides (LPS). The LPS from twenty strains of P. penneri were chemically analysed and their chemotypes discussed [3]. An unusual amino sugar has been found in P. penneri strain 16 LPS and identified as 3,6-dideoxy-3-(R)-3-hydroxybutyramido-D-galactose 1 [4]. In this paper we describe the structural elucidation of the 0-specific polysaccharide chain of this LPS together with some serological data.
MATERIALS AND METHODS
Miscellaneous
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