Immunochemical studies on Brucella abortus lipopolysaccharides

Abstract

The antigenic-toxic complex of B. abortus isolated in the phenol phase of phenol/water system, is a lipopolysaccharide (LPS)-protein macromolecule. The specific side chain was isolated from this complex by means of pronase treatment and mild HCl cleavage, followed by fractionation on Sephadex. The side chain thus isolated lost its capacity to coat erythrocytes but not that of neutralizing antibodies, as shown in the hemagglutination inhibition test. Esterification of the side chain with lauryl sulfat restored its erythrocyte coating capacity. In view of transforming this haptene into an immunogen, it was attached to S. minnesota Re cells. Immunization of rabbits with this conjugate engendered the formation of antibodies that agglutinated B. abortus and B. melitensis cells as well. As the side chain could only be isolated from the smooth strain, it proves that a defect in the synthesis of this moiety gave rise to the rough Brucella mutant. In the latter fraction glucose, mannose and quinovosamine were identified. Fraction II separated on Sephadex was identified as the core polysaccharide, present in the preparation obtained from smooth and rough strain. In the core region 2-keto-3-deoxy-octonate, glucose, glucosamine (from the smooth but not from the rough strain) and protein were identified. Following the mild HCl hydrolysis, the lipid-protein moiety i.e. region 3, is also split off but without precipitating and, during the fractionation on Sephadex, it adsorbs onto the gel. The absence of beta-hydroxymyristic acid and glucosamine in Brucella lipid dissociates it from the enteric lipid A. In the LPS-protein's toxicity a major role has the protein present only in the smooth strain.