Biomedical subjects
Publications and source records attributed to F San-Blas.
Chlamydospores and arthrospores are produced by the mycelial phase of Paracoccidioides brasiliensis in submerged cultures at 23 degrees C. Arthrospores are formed by extrusion in specialized, sporogenous hyphae. The absence of any unique morphological characteristic in chlamydospores and arthrospores discards the use of spore morphology as a taxonomic criterion.
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.
The approach to this article will be the review of experimental developments achieved in recent years in the fields of biochemistry, molecular biology, ultrastructure, etc. and that have suggested models and hypothesis in order to understand the regulation of the molecular mechanisms involved in fungal differentiation. Since fungal dimorphism has been seen by many investigators as a useful model of differentiation in eukaryotic systems, and also because of the prevalence of dimorphism among human pathogenic fungi, we have considered relevant to review the work done in Mucor, but also in dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, Histoplasma capsulatum, Candida albicans, Blastomyces dermatitidis, and Sporothrix Schenckii. From this point of view, the involvements of cyclic nucleotides, nucleic acid polymerases, synthesis of macromolecules, synthesis of cell wall and less studied factors will be reviewed.
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.
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.
A lethal diffusible metabolite excreted by the yeastlike form of Paracoccidioides brasiliensis was isolated, purified, and partially characterized. The chemical structure of this metabolite suggested a relation to the phenol or benzoic acid families. The lethal effect on P. brasiliensis could not be attributed specifically to interference with synthesis of macromolecules, but probably through protein denaturation. This lack of specificity discounts its possible use as an antibiotic.
Yeast and mycelial particulate preparations of Paracoccidioides brasiliensis were subjected to the action of several detergents in an attempt to solubilize the glucan synthetase present in these preparations. This was achieved more successfully in the yeast membranes than in the mycelial ones. The enzymatic activity was greatly stimulated in the insoluble fractions upon treatment with some of the detergents used. The results suggest that the yeast and mycelial phases of P. brasiliensis may differ in the structures of their membranes and also in the characteristics of their glucan synthetases.
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Spherical and osmotically sensitive protoplasts were released from cultures of the yeast-like form of Paracoccidioides basilienisis strain IVIC Pb9 through the action of a mixture of crude enzyme preparations: alpha and beta-glucanases and chitinase, obtained from culture filtrates of Cladosporium resinae, Basidiomycete QM 806 and Streptomyces sp respectively. The highest efficiency of protoplast liberation was achieved when each crude enzyme preparation was used at 1 mg/ml.
Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis or South American blastomycosis. Many aspects of the disease and its agent are unknown. One of the most important factors regarding the infection and the host-parasite relationships seems to be the fungal cell wall whose biochemical aspects are reviewed here. Biochemical studies, done mainly by Kanetsuna et al., (21,22), have demonstrated that the yeastlike (Y) and the mycelial (M) forms have chitin as a common polysaccharide, with alpha-1, 3-glucan in the Y form and beta-1, 3-glucan in the M form. These polysaccharides are fibrillar and determine to some degree the fungal shape. Moreover, an amorphous galactomannan is found in the cell wall of the M form. This compound is responsible for the antigenic properties of the cell wall (1). Recent studies (30-33) suggest that the cell wall does not possess a stable chemical structure but a rather changing one, as a function of the environment in which the fungus is grown. At the same time, the cell wall composition seems to correlate with the degree of virulence of the particular strain. From these observations it may be deduced that the constituent polysaccharides of P. brasiliensis cell wall, play an important role in the active protection of the fungus against the defensive mechanisms of the host.
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.
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.
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.
Growth curves of the yeast-like form of 4 strains of Paracoccidioides brasiliensis based on colony counts on GGY agar and on cell counts weree made. Mean generation times wer 17, 13 and 21 h. for 3 of the strains. The kinetics of growth for all strains showed that dead cells were present in approximately the same proportion as living cells in the initial inocula and their number increased through the exponential phase of growth at a similar rate as that of living cells. Viability counts made in 2-week old cultures of all strains were negative. It is suggested that during growth P. brasiliensis continuously diffuses a metabolite which is lethal to the fungus. Strains of Blastomyces dermatitidis and Histoplasma capsulatum were also sensitive to this lethal effects.