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The effect of fatty acids on Mycobacterium smegmatis was examined in vitro at pH 5.0 to 7.0 to determine the role of fatty acids in the intracellular killing of mycobacteria. Unsaturated fatty acids showed strong bactericidal activity in low concentrations (0.005 to 0.02 mM), whereas saturated fatty acids, except for lauric and myristic acids, were not very effective even at a concentration of 0.2 mM. Addition of a saturated fatty acid (palmitic or stearic acid) to an unsaturated fatty acid (oleic or linoleic acid) did not strongly interfere with the bactericidal effect of the unsaturated fatty acid at pH 5.0 and 6.0. Ca2+ (3.0 mM), Mg2+ (1.0 mM), and gamma-globulin (0.4%) showed weak reversal effects on the bactericidal activity of unsaturated fatty acids at pH 5.0 and 6.0. Serum albumin and serum showed strong reversal effects. The concentrations of each fatty acid in a mixture (molar ratio, 1:1:1:1) of oleic, linoleic, palmitic, and stearic acids required for the killing of M. smegmatis in the presence of 2% serum (bovine, rabbit, or human) were 0.05 to 0.10 mM at pH 5.0 and 6.0 and 0.05 to 0.20 mM at pH 7.0, depending on the serum used. The susceptibilities of M. kansasii, M. bovis strain BCG, and M. tuberculosis to the mixture of the four fatty acids in the presence of 2% bovine serum were similar to that of M. smegmatis, although M. fortuitum was more resistant.

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Whole cells or cell walls of the yeastlike and mycelial forms of Paracoccidioides brasiliensis, Blastomyces dermatitidis and Histoplasma capsulatum were treated successively with sodium hydroxide, beta-1,3-glucanase and pronase. The microfibrils in the insoluble residues, probably composed of chitin, were examined in the electron microscope. In the yeastlike form, tightly interwoven, randomly oriented microfibrils were seen. On the other hand, in the mycelial form, a large portion of microfibrils tended to lie in a more or less longitudinal orientation. A role of chitin in the conversion from the yeastlike form to the mycelial form is discussed.

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The effect of lysozyme on the growth of several strains of mycobacteria was examined at pH 5.0-7.0 in Dubos medium containing various concentrations of lysozyme (100-2,000 microgram/ml). Mycobacterium smegmatis and M. phlei were susceptible to lysozyme at pH 5.0-7.0. The effect of lysozyme was marked between pH 6.0 and 7.0 and the colony counts were reduced to approximately 0.1-10% after incubation with 100 micrograms of lysozyme per ml for 48 hr. At pH 5.0, 10-40% of the organisms survived treatment with 1,000 micrograms of lysozyme per ml for 48 hr. M. bovis strain BCG, M. tuberculosis, and M. fortuitum appeared to be more resistant to lysozyme than M. smegmatis and M. phlei. M. smegmatis and M. phlei did not contain detectable amounts of poly-L-glutamic acid, although the susceptibility of the mycobacteria to lysozyme did not correlate with the amounts of the polymer in the cell walls. The role of lysozyme in animal infections with so-called saprophytic mycobacteria is discussed.

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The adjuvant activity of cell wall skeletons (mycolic acid-arabino-galactan-mucopeptide, CWS) prepared from the cells of mycobacteria, nocardia and corynebacteria was examined in vivo in mice and guinea pigs. The cell wall skeletons of Mycobacterium bovis BCG (BCG-CWS), Nocardia asteroides 131 and Corynebacterium diphtheriae PWC suspended in Freund's incomplete adjuvant (FIA) as water-in-oil emulsions showed potent adjuvant activity on the formation of circulating antibody and cell-mediated immunity to bovine serum albumin (BSA), sheep erythrocytes (SRBC) and sulfanylazo-bovine serum albumin (SA-BSA) in mice and guinea pigs. After acetylation or acid treatment, BCG-CWS retained its adjuvant activity, but the activity of BCG-CWS was destroyed completely by alkaline treatment. The cell wall constituents, arabinose-mycolate and arabino-galactan, prepared from BCG-CWS showed no adjuvant activity. It was also shown that BCG-CWS suspended in phosphate buffered saline or associated with oil droplets augmented cell-mediated cytotoxicity in which thymus-derived lymphocytes (T-cells) are effector cells.

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The biochemical and morphological changes of the yeastlike (Y) form to the mycelial (M) form of Paracoccidioides brasiliensis were examined. The main polysaccharide of hexoses of the Y-form cell wall was alpha-glucan, whereas the polysaccharides of the M-form cell wall were beta-glucan and galactomannan. The alpha-glucan of the Y form contained mainly alpha-(1 --> 3)-glycosidic linkage. The beta-glucan of the M form contained mainly beta-(1 --> 3)-glycosidic linkage with a few branches at C-6 position. The incorporation of (14)C-glucose into the cell wall glucans showed that synthesis of alpha-glucan decreased rapidly after the temperature of the culture was changed from 37 to 20 C. The synthesis of beta-glucan was augmented at an early stage of the morphological change. The M-form cell wall contained 12 times more disulfide linkage than the Y form. The cell-free extracts of the whole cell of the Y form had five times more protein disulfide reductase activity than the M form, whereas extracts of the M form contained five to eight times more beta-glucanase activity than the Y form. From these results, a hypothesis for the production of the M form from the Y form is proposed.

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The thermally induced changes in the cell wall polysaccharides of Blastomyces dermatitidis strain BD64, which produces a yeastlike form (Y form) at 37 C and a mycelial form (M form) at 20 C, were examined. The cell walls of the Y and M forms contained 36 and 51% of hexoses, respectively. The M-form cell wall contained glucose, galactose, and mannose in a molar ratio of 1:0.1:0.2. The Y-form cell wall contained mainly glucose and a very small amount of galactose and mannose. The glucans of the cell wall of the Y form consisted of about 95% alpha-glucan and 5% beta-glucan, whereas those of the M-form cell wall consisted of about 60% alpha-glucan and 40% beta-glucan.

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Glucans were isolated from the cell wall of the yeast (Y) and mycelial (M) forms of Paracoccidioides brasiliensis. The alkali-soluble glucan of the Y form had properties of alpha-1,3-glucan. The alkali-insoluble glucan of the M form was identified as a beta-glucan which contains a beta-(1 --> 3)-glycosidic linkage by infrared absorption spectrum, by effect of beta-1,3-glucanase, and by partial acid hydrolysis. The alkali-soluble glucans of the M form were a mixture of alpha- and beta-glucans and the ratio of alpha- to beta-glucan was variable, depending on the preparations.

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Short and thick fibers were observed on the outer surface of the yeast phase of Paracoccidioides brasiliensis, and long and thin fibers were seen on the inner surface. The long fibers disappear with chitinase treatment and are composed of chitin. The short fibers disappear under alkali treatment and are composed of alpha-glucan. Comparisons with alpha-(1 --> 3)-glucan isolated from Aspergillus niger and Polyporus betulinus and with chitin from fungal origin support our point of view.

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