Search

The total lipid extracted from the yeast form of Paracoccidioides brasiliensis was twice as much as from the mycelial form. In both morphological forms neutral lipids were predominant and triglycerides were the greatest single component in the extracts. Relative percentage of phospholipid was higher in Y form than in M form. Phosphatidylcholine was predominant in the former and phosphatidylethanolamine in the latter. Diphosphatidylglycerol was the most unsaturated lipid in both the forms, and in the Y form it was the only individual class of lipid that contained a greater percentage of linoleic than oleic acid. Otherwise, the lipids of Y form, both neutral and polar, were characterized by a greater percentage of oleic acid and those of the M form by the predominance of linoleic acid. Differential distribution of phosphatidylcholine, phosphatidylethanolamine, sterol, oleic and linoleic acid in Y and M forms is interpreted to suggest a possible role of cellular lipid composition in the dimorphic behaviour of P. brasiliensis.

Differences in cell wall structure of Paracoccidioides brasiliensis strains isolated from localized or disseminated cases of paracoccidioidomycosis and from soil emphasize the importance of standardization if results obtained in different laboratories are to be compared.

A peptido-polysaccharide isolated from the culture medium of Paracoccidioides brasiliensis has been studied. The crude product is a mixture of several polysaccharides releasing, by hydrolysis glucose, mannose and galactose. The purified fraction contains mannose (88%) and galactose (12%). This galactomannan has a molecular weight varying between 4.6.10(4) and 2.6.10(4). It is associated with a peptide containing four amino-acids.

The lipid content and composition of four strains of Paracoccidioides brasiliensis were analysed to determine any possible correlation with their virulence for hamsters and mice. Two strains, Pb168 and Pb141, were equal in virulence, Pb9 was slightly virulent and Pb140 was avirulent under the experimental conditions. No correlation was observed between virulence and the total lipid or phospholipid content of the strains. The lipid yield was highest in Pb9 and lowest in Pb168. Polar lipids were highest in Pb9 and least in Pb140. Phosphatidylcholine was the dominant phospholipid in all strains but its percentage was lower in the avirulent strain Pb140. Diphosphatidylglycerol, the least saturated lipid in all strains, was less abundant in Pb140 than in the virulent strains Pb168 and Pb141. In all four strains, neutral lipids constituted the major fraction of total lipids and triglycerides were the predominant individual lipid class, being more abundant in the avirulent and slightly virulent strains that in the virulent strains. The fatty acid profiles of total lipids and individual lipid classes of neutral and polar lipids obtained from the four strains were similar; however, the individual lipid classes showed patterns of preferential distribution of these fatty acids.

Nitrosoguanidine treatment of Paracoccidioides brasiliensis strain IVIC Pb73 induced a set of mutants affected in their ability to complete dimorphism. Some did not transform to the typical mycelial form, while others did not grow as yeasts. These mutants were preliminarily characterized by microscopic observations and the study of some of their properties: optimal growth temperature, nutritional requirements, virulence and cell wall analyses. Comparison of these properties allowed the classification of the mutants in 4 functional categories with respect to dimorphism.

The occurrence of a polysaccharide fraction of Paracoccidioides brasiliensis cell wall with toxic, granuloma-inducing and macrophage-stimulating activities was demonstrated. After fractionation of the lipid-extracted wall with 1 M-NaOH, three fractions were obtained: (1) an alkali-insoluble fraction; (2) an alkali-soluble, acid-insoluble fraction and (3) an alkali-soluble, acid-soluble fraction. When the three fractions were injected into mice only fraction (1) was able to induce chronic lung inflammation, causing a marked loss in body weight and death at a dose of 6 mg per animal. Analysis of the stimulation of peritoneal macrophages of mice (measured by cell spreading on glass) after intraperitoneal injection of fraction 1 showed that 75% of the cells were able to spread even 20 d after inoculation.

Yeast cells of Paracoccidioides brasiliensis strain SN, were obtained for analysis of lipid composition. Total lipids, phospholipids, sterols, and qualitative sterols and fatty acid composition were determined. Such analysis were made on cells cultured in the presence or absence of amphotericin B and on non proliferating cell suspensions exposed to the antibiotic. Marked alterations in lipid contents were observed in this different conditions. The major alterations were the reduction of total lipids, sterols, and palmitoleic acid in both, proliferating and non proliferating antibiotic exposed cells. The effect of amphotericin B was evaluated also in terms of viability and release of intracellular substances, at different times of exposure. The minimal inhibitory concentration (MIC) determined for that strain of this fungus was 0.2 microgram/mL.

An adenine-requiring mutant of Paracoccidioides brasiliensis strain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Cell wall analysis of this mutant (strain IVIC Pb141) showed an increase in the amount of alpha-1,3-glucan and a virtual disappearance of the antigenic galactomannan. At the same time, a higher degree of virulence was observed for the mutant. These results agree with the hypothesis presented before (16) about a relationship between cell wall polysaccharides and pathogenicity in P. brasiliensis.

A morphological mutant of Paracoccidioides brasiliensis strain IVIC Pb9 was isolated after treatment of the yeast-like (Y) form with nitrosoguanidine. Colonies of the mutant grown at room temperature did not show the whitish cotton-like morphology typical of the mycelial form of the parental strain. Y-cells were much smaller than those produced by the parent and grew forming chains of different sizes. The main chemical difference in the wall of the Y-form was the replacement of the alpha-I,3-glucan, typical of the parental strain, by an amorphous I,3-mannan in the mutant.

The yeastlike form of Paracoccidioides brasiliensis strain IVIC Pb9 reduced the amount of alpha-1,3-glucan in its cell wall from 45 to 3% when subcultured in vitro for several years. This strain regained its alpha-1,3-glucan up to 25% of the total cell wall when grown in vivo. A mutant strain of P. brasiliensis Pb9, named IVIC Pb140, reported to have 1,3-mannan instead of alpha-glucan in the cell wall, could not be recovered from experimentally infected animals. The existence of some relationship between the presence of alpha-1,3-glucan in the cell wall of the yeastlike form and the pathogenicity of this fungus is suggested in this report.

We carried out a comparative study of the histopathology (lung, liver, spleen, kidney and adrenals) and the anti-P. brasiliensis humoral (immunodiffusion test) and cellular (footpad test) immune response of mice intravenously inoculated with yeast forms of three P. brasiliensis isolates (Pb 18, Pb 192, Pb 265). Pb 265 (avirulent strain) did not evoke specific lesions or antibody production; the levels of cellular immunity were significantly lower than those of the two other isolates. Lung granulomas induced by strain Pb 18 were richer in fungi and neutrophils and poorer in mononuclear cells when compared to those induced by strain Pb 192. Extrapulmonary lesions were more frequent in mice infected with strain Pb 18. Strains Pb 18 and Pb 192 raised similar humoral and cellular anti-P. brasiliensis responses. Cell wall analysis did not suggest striking differences among the strains. Slightly higher levels of the water soluble fraction 3 (which contains the immunogenic galactomannan and protein) were detected in strain Pb 265.

A human isolate (IVIC Pb73) of paracoccidioides brasiliensis and a mutant strain (IVIC Pb219) derived from Pb73 were studied with regard to their cell wall composition. Differences were found between these strains and the only other strain (IVIC Pb9) previously studied from this point of view. Besides the expected beta-1,3-linkages, beta-1,2-bonds were also found in the alkali-insoluble glucan of both the parental and the mutant strains in the yeast forms, a rather uncommon linkage in fungal glucans. Also, the alkali-insoluble fraction from the mycelial form was composed of glucose (of which a very small proportion occurred as beta-1,3-glucan) plus a high proportion of galactose (about 50%), and some mannose. The alkali-soluble fraction, usually associated with the immunogenic galactomannan in the mycelial form, also had glucose besides galactose and mannose, but was exclusively composed of mannose in the yeastlike form. These results suggest that, as in other fungi, variations in cell wall composition could be expected between strains of P. brasiliensis.