Fractionation of mycobacterial cell wall. Isolation of arabinose mycolate and arabinogalactan from cell wall fraction of Mycobacterium tuberculosis strain Aoyama B.

In previous papers, we reported the isolation and characterization of arabinose mycolate from firmly bound lipids (7; Azuma and Yamamura, in preparation) and wax D (3) fractions of Mycobacterium tuberculosis and other mycobacterial strains. The chemical structure of arabinose mycolate has also been established as D-arabinose5-mycolate (7). The serologically active polysaccharide, arabinogalactan, was purified from defatted cells (2), culture filtrate (4), and wax D fraction (5) of M. tuberculosis strain Aoyama B in our laboratory. We have suggested that arabinose mycolate and arabinogalactan were the principal components of mycobacterial wax D and defatted cells. In this note, we describe fractionation of lipid fractions from the cell wall and isolation of arabinose mycolate and arabinogalactan from the cell wall of M. tuberculosis. The chemical structure of the mycobacterial cell wall is discussed. The cell wall fraction was prepared by the following procedure. Acetone-dried M. tuberculosis cells were suspended in cold saline to a concentration of 100 mg (dry weight) per ml. This suspension was then processed in a Sorvall Ribi refrigerated cell fractionator at 25,000 lb/in2 at 5 to 10 C. The effluent was centrifuged at 800 X g to remove unbroken whole cells. The supernatant fluid was centrifuged at 15,000 X g for 60 min to obtain a supernatant fluid containing most of the soluble cytoplasm and a sediment consisting predominantly of cell wall. The supernatant fluid was recentrifuged at 40,000 x g for 2 hr to obtain a sediment consisting of reddish particles. The supernatant fluid was recentrifuged at 100,000 x g for 2 hr to yield a small amount of sediment consisting of smaller particles. The crude cell wall fraction was suspended in water and centrifuged at 800 x g to remove unbroken whole cells again. After washing in water three times, the cell wall fraction was lyophilized. The yield of the lyophilized cell wall fraction was approximately 10% of starting cells. The cell wall fraction had a characteristic surface structure of paired fibrous prominence by electron micrographic examination. An 8-g amount of cell wall fraction thus purified was fractionated into 11 lipid subfractions by the method of Anderson (1), with some modifications in the extraction of firmly bound lipids (6). Table 1 shows the results of lipid fractions from the cell wall and intact cells. Larger amounts of lipid were contained in the cell wall fraction than intact cells. Tuberculin-active protein was also obtained from the cell wall ofhuman tubercle bacilli. The chemical and immunological properties of tuberculin-active protein will be reported in a later paper. Arabinose mycolate was purified from the firmly bound lipid B fraction of the cell wall of M. tuberculosis by the column chromatographic procedure. A 700-mg amount of the firmly bound lipid B fraction, dissolved in n-hexane, was loaded on a column of 50 g of silicic acid-Celite (2:1) and eluted with chloroform and chloroform-methanol (95:5, 9:1, and 8:2). A 400-mg amount of the second fraction eluted with chloroform-methanol (95:5) in chromatogram I was rechromatographed on a column of 20 g of Florisil-Celite (2:1) and eluted with ether and ether-methanol (95:5, 9:1, and 8:2). The second fraction, eluted with ether-methanol (95:5) in chromatogram II, was dissolved in ether and precipitated with methanol, and 210 mg of white powdered lipid fraction was obtained. The physicochemical properties of this lipid fraction are shown in Table 2. The infrared spectrum of this lipid fraction was almost identical with that of D-arabinose-5-mycolate established previously (7). By acid or alkaline hydrolysis with a 5% HCI or 1% NaOH solution, D-arabinose and mycolic acid were obtained. Mycoloyl alcohol was obtained by the reductive cleavage with LiAlH4. Mycolic acid and mycoloyl alcohol were determined by infrared spectroscopy and elementary analysis. D-Arabinose was determined by gasliquid chromatography (2). By the quantitative determination of arabinose after acid hydrolysis