Chemical and Immunological Studies on Mycobacterial Polysaccharides I. Purification and Properties of Polysaccharides from Human Tubercle Bacilli

Abstract

Defatted human tubercle bacilli, Aoyama B strain, were extracted with 0.1 n NaOH for 24 hr, and the crude polysaccharide fraction was precipitated by the addition of 5 volumes of ethyl alcohol. A yield of 17.8 g of crude polysaccharides was obtained from 800 g of bacilli. The crude polysaccharide was further fractionated into seven fractions by fractional precipitation with ethyl alcohol. Each fraction was purified by successive chromatography on Dowex 50 and diethylaminoethyl cellulose, and by gel filtration on Sephadex G-75 and G-200. Optical rotation and gas chromatographic analyses of purified polysaccharide showed that these polysaccharides contained glucan mannan, arabinomannan, and arabinogalactan. Each polysaccharide was almost completely free from nitrogen, and no tuberculin reaction was produced by 100 μg of each material. Arabinomannan and arabinogalactan showed precipitin reaction, complement fixation, and passive hemagglutination reaction with rabbit antiserum against heat-killed whole bacilli (Aoyama B). In guinea pigs sensitized with Aoyama B bacilli, arabinomannan and arabinogalactan provoked anaphylactic shock when injected intravenously, and Arthus type reaction when injected intracutaneously. With the use of rabbit antiserum, arabinomannan and arabinogalactan showed passive anaphylactic shock, passive cutaneous anaphylaxis, and Prausnitz-Küstner type reactions in guinea pigs. By immunodiffusion analysis, it was shown that the antigenic determinant of arabinomannan was different from that of arabinogalactan.