Purified native haptens of Brucella abortus B19 and B. melitensis 16M reveal the lipopolysaccharide origin of the antigens

Abstract

Purification of the Brucella polysaccharide referred to as native hapten (NH) and extracted from cells by the autoclaving procedure, was accomplished by ultrafiltration, followed by repetitive gel filtration using high-performance liquid chromatography on a ⪡TSK-G2000-SW⪢ column. The purified NH was analysed by SDS-PAGE, gas-liquid chromatography mass spectroscopy, and 13 C and 1 H NMR spectroscopy. NH from B. abortus B19 (NH-A) was shown to have a structure identical to that of A polysaccharide from B. abortus 1119-3, a linear homopolymer of α-1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues. The structure of the NH from B. melitensis 16M (NH-M) was identified as a linear homopolysaccharide of the same sugar but composed of a pentasaccharide repeating unit in which four α1,2-linked-4,6-dideoxy-4-formamido-D-mannopyrannosyl residues are linked α-1,3 to the last monosaccharide of the sequence. This structure is similar to that determined for the Brucella M polysaccharide from B. melitensis 16M. The discovery in highly purified NH preparations of covalently bound monosaccharides characteristics of lipopolysaccharide inner core regions e.g. , quinovosamine, mannose and 3-deoxy-D-manno-octulosonate (KDO), indicates that this polysaccharide is derived from lipopolysaccharides (LPS) by hydrolytic conditions fortuitously generated during the extraction protocol. The antigenically important polysaccharides of Brucelle are now established to be either A or M antigens. Polysaccharide B is a cyclic glucan with no structural or serological relationship were to A for M polysaccharides, its apparent activity in diagnostic tests of infected cattle resuls from O polysaccharide B.