Determination of the growth curves of the mycelial and yeast forms of Paracoccidioides brasiliensis

Summary The growth curves of 2 different strains ofP. brasiliensis were determined. Cultures were made in a dialyzed trypticase soy broth and the growth assesed at selected intervals by means of viable cell counts. In the yeast form, both strains exhibited an uniform pattern of growth; the mycelial forms were also similar although one of the strains survived for a longer period of time. When the 2 yeast curves were compared with the mycelial ones, large differences were noticed. Thus, the mean survival time for the yeasts was 13.5 days while it was 50.25 days for the mycelia. During the exponential phase, the mean rate of growth was accelerated for the yeasts (0.110) in comparison with the one exhibited by the mycelia (0.0265).Irrespective of the form of growth and for both strains, death occurred abruptly, a few hours after a period when colony counts were high, indicating that the fungus was viable at the preceeding interval.This paper includes, in part, a thesis submitted by M. Arango to the University of Antioquia, Medellín, in fulfillment of the requirements for the Master of Science degree.     

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.     

Growth curves,morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 10⁶ FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×10⁶ FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×10⁶ FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.     

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.     

Liberation of protoplasts from the yeast-like form of Paracoccidioides brasiliensis by enzymatic treatment

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.     

Characteristics of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The sporulation capacities of the mycelial form of Paracoccidioides brasiliensis were determined. Five different culture media were used and four human isolates studied. Conidia were produced in three agar media, namely water-agar, glucose-salts and yeast-extract. Corn meal and Sabouraud dextrose agars failed to induce sporulation. Various types of spores were characterized with peculiar bulging arthroconidia and single-celled, pear-shaped conidia predominating. The size of these conidia varied from 3.6 to 4.6 micron in length. It is concluded that the mycelial form of P. brasiliensis produces characteristic spores if the proper culture media are employed.     

Ultrastructural changes produced by ketoconazole in the yeast-like phase of Paracoccidioides brasiliensis and Histoplasma capsulatum

The ultrastructural changes produced by ketoconazole on the yeast-phase ofH. capsulatum andP. brasiliensis were studied by means of scanning and transmission electron microscopy.The observed alterations on both fungi were very similar to those induced by the same drug on the ultrastructure ofC. albicans. These alterations include surface changes, abnormal membrane proliferation, fatty degeneration of the cytoplasm and lysis of subcellular organelles. P. brasiliensis seems to be more sensitive to ketoconazole thanH. capsulatum, since the necrosis of most of the cells was obtained in the former at a concentration of 0.1 gmg/ml and in the latter at 1 g/ml.     

Small forms and hyphae of Paracoccidioides brasiliensis in human tissue

A 53 year-old man had a three-year recurrent respiratory infection. No fungi was detected in sputum examinations. Immunodiffusion test with paracoccidioidin revealed two precipitin bands. Very small forms and hyphae of a fungus were seen on silver methenamine stained serial sections from lung's lesion. P. brasiliensis was identified on the basis of the rare multibudding small forms.     

Scanning electron microscopy of the conidia produced by the mycelial form of Paracoccidioides brasiliensis

The conidia produced by the mycelial form of Paracoccidioides brasiliensis were examined by scanning electron microscopy for the first time. Several different conidial types were characterized. These included intercalary arthroconidia, several types of septate conidia that are formed from other conidia, pedunculate conidia, and terminal hyphal conidia. In addition, the ultrastructure of the supporting pedestal of the pedunculate conidium was found to be separated from the mother conidium by a septum in some instances, and at other times it was not.     

Aleuriospores of Paracoccidioides brasiliensis

In soil extract agar and in Bennett medium abundant aleuriospores ofParacoccidioides brasiliensis have been observed. The possibility that these spores could be the infecting elements for man is discussed.

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Resumen Se han observado abundantes aleuriosporas deParacoccidioides brasiliensis en agar extracto de tierra y en medio de Bennett. Se discute la posibilidad de que éstas esporas puedan ser los elementos que infectan al hombre.

     

The pathobiology of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis. It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form. This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall. Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response. Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males. Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production. The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P. brasiliensis existing as a facultative intracellular pathogen.     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998