Immunological studies on Histoplasma capsulatum.

Alveolar macrophages freshly harvested from normal and immunized rabbits were parasitized with yeast cells and protoplasts of Histoplasma capsulatum. Macrophages obtained from either normal or sensitized rabbits failed to phagocytize protoplasts, whereas, the yeast cells were actively ingested. There was no detectable intracellular killing by macrophages. A serological similarity was found between the whole yeast cell, the purified isolated cell wall, and the protoplasts of the fungus. Aprecipitin test of the protoplasts of the fungus gave a postive band, whereas immunodiffusion in agar was negative. Addition of immune sera activated phagocytosis, the immune sera against cell walls being the most active.

Chemical and ultrastructural studies on the cell walls of the yeastlike and mycelial forms of Histoplasma farciminosum.

The cell wall of the yeast form of Histoplasma farciminosum contains 13.2% beta-1,3-glucan, 1.0% galactomannan, and 25.8% chitin, whereas the cell wall of mycelial form has 21.8, 4.5, and 40%, respectively, for the same polymers. Also, the cell wall of the yeast form contains alpha-1,3-glucan (13.5%) and an unidentified polymer (21.5%). Chitin, one of the structural polymers of both yeast and mycelial cell walls, is identified as thin isolated fibers (4 nm wide) or in thick bundles (50 nm wide) of fibers. beta-(1-3)-Glucan is also found as thin isolated fibers indistinguishable from isolated fibers of chitin. Fibers 14 nm wide and resembling alpha-(1-3)-glucan fibers of other fungi are found in the yeast form. The results reported here do not give support to the proposal for a different taxonomic classification.

Kinetic and morphological observations on the yeast phase of Histoplasma capsulatum during protoplast formation.

Protoplast formation by Histoplasma capsulatum yeasts using high concentrations of MgSO(4) occurs either by lysis of the bud or lysis of the entire cell wall. Both mechanisms may also occur simultaneously. Neither the protoplast emerging through a hole in the cell wall nor the freshly released protoplast has a recognizable cell wall or the remnant of such. The protoplast contains all the organelles of the normal cell except for mesosomes. During protoplast formation the nucleus increases in size and produces several nuclear masses by the invaginations of the internal layer of the nuclear membrane. All these nuclear masses are surrounded by the external layer of the nuclear membrane. Several nuclei with a normal nuclear membrane are formed later.

Biochemical studies on the thermal dimorphism of Paracoccidioides brasiliensis.

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.

Cell wall composition of the yeastlike and mycelial forms of Blastomyces dermatitidis.

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.

Cell wall glucans of the yeast and mycelial forms of Paracoccidioides brasiliensis.

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.

Chemical morphology of glucan and chitin in the cell wall of the yeast phase of Paracoccidioides brasiliensis.

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.

Ultrastructure of dimorphic transformation in paracoccidioides brasiliensis.

The fine structure of Paracoccidioides brasiliensis undergoing temperature-dependent transformation from mycelium to yeast and vice versa (M right harpoon over left harpoon Y) was studied. The transitional form to mycelium from the yeast appears as an elongated bud that extends from the yeast and which has a mixture of characteristics from both the yeast and the mycelium. The transitional form to yeast from the mycelium starts with enlargement of the interseptal spaces and cracking of the outer electron-dense layer of the cell wall of the hypha. Later the interseptal spaces tend to become round and separate. In M --> Y only few interseptal spaces seem to transform. The yeast is produced by self-transformation of the hypha. In Y --> M a new structure is formed and the yeast dies. Intrahyphal hyphae are observed during the transformation from M --> Y, and intrayeast hyphae during the Y --> M. Due to the high mortality and breakage observed in both types of transformations, we believe that wound of the yeast or the mycelium could elicit this phenomenon.

Intrayeast hyphae in Paracoccidioides brasiliensis.

The occurrence of a live hypha inside a dead yeast during the transformation from yeast to mycelium of Paracoccidioides brasiliensis is described.

Cell wall composition of the yeast and mycelial forms of Paracoccidioides brasiliensis.

Isolation and chemical analyses of the cell walls of the yeast (Y form) and mycelial forms (M form) of Paracoccidioides brasiliensis and Blastomyces dermatitidis revealed that their chemical composition is similar and depends on the form. Lipids, chitin, glucans, and proteins are the main constituents of the cell walls of both forms of these fungi. There is no significant difference in the amount of lipids (5 to 10%) and glucans (36 to 47%) contained by the two forms. In both fungi, the Y form has a larger amount of chitin (37 to 48%) than the M form (7 to 18%), whereas the M form has a larger amount of proteins (24 to 41%) than the Y form (7 to 14%). Several properties of the glucan of P. brasiliensis were studied. Almost all of the glucan in the Y form was soluble in 1 n NaOH, was weakly positive in the periodic acid-Schiff reaction, was not hydrolyzed by snail digestive juice, and had alpha-glycosidic linkage. Glucans of the M form were divided into alkali-soluble (60 to 65%) and alkali-insoluble (35 to 40%) types. The alkali-soluble glucan was similar to that of the Y form; the alkali-insoluble glucan was positive in the periodic acid-Schiff reaction and was hydrolyzed by snail digestive juice.

Mycelial phase of Paracoccidioides brasiliensis and Blastomyces dermatitidis: an electron microscope study.

A comparative study of the mycelial phase of Paracoccidioides brasiliensis and Blastomyces dermatitidis reveals that both fungi are very much alike, containing multiple nuclei and nuclear pores, mitochondria, ribosomes, scarce endoplasmic reticulum, intracytoplasmic membrane systems, glycogen, and vacuoles. Shadowed cell walls show fine fibrillar surfaces that contrast with those in the yeast phase. The intracytoplasmic membrane system is continuous with the plasma membrane and is similar to bacterial mesosomes. Granules with light cores and dark rims are observed in the plasma membrane. Live hyphae growing inside a dead hypha are found much more frequently in immersed cultures than in solid-medium cultures, suggesting that breakage of the hypha could elicit this phenomenon.

Cell wall changes during the budding process of Paracoccidioides brasiliensis and Blastomyces dermatitidis.

The difference between the budding process of Paracoccidioides brasiliensis and Blastomyces dermatitidis is reported herein. A characteristic feature in P. brasiliensis is that the optical density of the cell wall increases at the site where budding begins and at the neck of the dividing cell, whereas B. dermatitidis does not undergo this alteration. The neck which is formed between the mother and daughter cell at the site of division is much wider in B. dermatitidis than in P. brasiliensis. The bud scar in P. brasiliensis appears as a truncated cone, the top of which is covered only by the inner layer of the cell wall; in comparison, in B. dermatitidis the bud scar exhibits a flattened surface covered by the cell wall. Both fungi show an increase in the number of mitochondria and infoldings of the cytoplasmic membrane at the site of separation, which indicates that at this site there is an increase of metabolic activity.

Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis.

Kanetsuna, Fuminori (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela), and Luis M. Carbonell. Enzymes in glycolysis and the citric acid cycle in the yeast and mycelial forms of Paracoccidioides brasiliensis. J. Bacteriol. 92:1315-1320. 1966.-Enzymatic activities in glycolysis, the hexose monophosphate shunt, and the citric acid cycle in cell-free extracts of the yeast and mycelial forms of Paracoccidioides brasiliensis were examined comparatively. Both forms have the enzymes of these pathways. Activities of glucose-6-phosphate dehydrogenase and malic dehydrogenase of the mycelial form were higher than those of the yeast form. Another 15 enzymatic activities of the mycelial form were lower than those of the yeast form. The activity of glyceraldehyde-3-phosphate dehydrogenase showed the most marked difference between the two forms, its activity in the mycelial form being about 20% of that in the yeast form.