Chemical composition of antigen 60 from Mycobacterium bovis BCG

Antigen 60 (A60), the main thermostable immunogen of tuberculin and PPD, has been purified from Mycobacterium bovis BCG cytoplasm, and identified by crossed immunoelectrophoresis with anti-BCG polyclonal antiserum. Two A60 fractions, free lipids and lipid-conjugated compounds, have been recognized. The free lipids represented about 30% (dry weight), and consisted essentially of C16-C18 fatty acids, and of phosphatidyl-inositol-mannosides. Lipoconjugates, upon DEAE-cellulose chromatography and gel filtration, yielded two main fractions of neutral and polar components. Chromatography of delipidated and deproteinized A60 on Sephadex G-100 yielded: a high molecular weight fraction (Al, 18%, a lipoglucan of congruent to 10(6)), and a low molecular weight fraction (B, 10%, a lipopeptidoglycan of congruent to 10(4)) containing mannose, glucose, and small amounts of arabinose. The polysaccharide moieties of fractions Al and B were submitted to acetylation, methylation, and acid hydrolysis, and the structure of the hydrolysed polymer was deduced by combined gas chromatography/mass spectrometry analysis. The results indicated a branched structure involving 1,4-, 1,6-, and 1,4,6-linked D-gluco- or D-manno-pyranosyl residues. Glucan- and peptidoglycan-bound fatty acids were identified as saturated (C16-C18) and monounsaturated linear acids (C12-C18). Immunodiffusion on agarose gel indicated that delipidation and proteolysis did not suppress the ability of A60 to yield immunoprecipitates with anti-A60 antiserum. The high polymer fractions obtained by chromatography on DEAE cellulose and Sephadex G-100 were also reactive. It is concluded that A60 is made of free lipids and of lipopeptidoglycans of high molecular weights (10(6)-10(7)) endowed with immunogenic properties.