RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis.

Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America. The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated. Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders. These results underline the belief that P. brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment.

Lysis of Paracoccidioides brasiliensis by Zygosporium geminatum.

Zygosporium geminatum, isolated as a contaminant in a culture of the mycelial phase of Paracoccidioides brasiliensis, was lethal to the latter organism. Its lytic action was due to exocellular alpha-1,3- and beta-1,3-glucanases which degraded the P. brasiliensis cell wall. The alpha-1,3-glucanase was more active at 30 degrees C and the beta-1,3-glucanase at 23 degrees C, each having pH 6.0 as its optimum.

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 degrees 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.

Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis.

Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of P. brasiliensis.

Polyamines in growth and dimorphism of Paracoccidioides brasiliensis.

Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine). Conjugated and bound polyamines were also quantified. 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P. brasiliensis.

Ornithine decarboxylase in Paracoccidioides brasiliensis.

Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37 degrees C in the yeast phase and at 30 degrees C in the mycelial phase. In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P. brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone. The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition.

Cladosporium carrionii and Hormoconis resinae (C. resinae): cell wall and melanin studies.

Two phaeoid strains of the fungus Cladosporium carrionii (SR3 from a xerophyte species and PP8201 from a patient), and one strain of Hormoconis resinae (Cladosporium resinae), isolated from oil-impregnated soil, were analyzed for their cell wall composition by colorimetric methods, X-ray diffraction, infrared spectroscopy, and solid-state 13C-nuclear magnetic resonance. Results suggested that the cell walls were composed mainly of hexoses (34%-47%) as beta-1,3-glucan (some galactose and mannose were also present) and melanin, chitin being absent. Electron microscopic observations suggested that melanin was found not only in the cell wall but also in intracellular bodies resembling melanosomes.

Production and regeneration of protoplasts from the Y-phase of the human pathogenic fungus Paracoccidioides brasiliensis.

Protoplasts of Y-cells and partially converted M-cells from several human isolates of Paracoccidioides brasiliensis were obtained with a combined enzyme system containing Novozym 234 and a chitinase. A laboratory made extract from Trichoderma harzianum CBS-345-33 supplemented with chitinase induced the release of protoplasts from partially converted M-cells but not from established Y-cells. A similar yield of protoplasts (1-2 x 10(5) ml-1 after 16 h) was produced by using either enzymatic system. Protoplasts regenerated on nutrient gelatin at 23 degrees C at a frequency of 0.1%.

Effect of ajoene on dimorphism of Paracoccidioides brasiliensis.

Ajoene, a compound derived from ethanolic garlic extracts, inhibits the growth of the dimorphic pathogenic fungus Paracoccidioides brasiliensis, yeast cells being more sensitive to its action than mycelial cultures. Sulphydryl protector groups were able to cancel the inhibitory effect on the yeast cells but not on the mycelial cultures. The dimorphic process was also blocked when mycelia were placed to transform to yeast. Synthesis in vitro of cell wall glucan was not affected by ajoene. We discuss the possible involvement of ajoene on sulphydryl metabolism of P. brasiliensis.

A preliminary study of in vitro antibiotic activity of saperconazole and other azoles on Paracoccidioides brasiliensis.

Saperconazole, a new triazole related to itraconazole, was tested against Paracoccidioides brasiliensis and results compared with ketoconazole and itraconazole. The fungus was highly sensitive to the action of these compounds, particularly saperconazole, with minimum inhibitory concentrations and minimum fungicidal concentrations ranging from 10(-7) to 10(-10) M (equivalent to 6.7 x 10(-2)-6.7 x 10(-5) micrograms ml-1 for saperconazole), according to the morphological phase and the antifungal tested. The yeast phase was more sensitive than the mycelial phase to any of the azoles. Morphological changes were observed in the cell membranes, particularly when saperconazole was used as the antifungal agent.

Mutants of Paracoccidioides brasiliensis strain IVIC Pb9 affected in dimorphism.

Morphological mutants were isolated after nitrosoguanidine treatment of Paracoccidioides brasiliensis strain IVIC Pb9. Two of these mutants, Pb257 and Pb258, developed a typical mycelia at 23 degrees C, however, the yeast cells which developed at 37 degrees C were indistinguishable from those of the parental strain. A third mutant, strain Pb267, was thermosensitive, grew as yeast-like cells at 23 degrees C, but was unable to survive at 37 degrees C. Morphological observations as well as serological and segregation tests confirmed that the mutant strains originated from P. brasiliensis. Cell wall chemical analyses of the mutant strains grown at 23 degrees C indicated the presence of alkali-soluble, acid-insoluble polysaccharides absent in the parental wild-type strain Pb9 grown under the same conditions. The phenotypes shown by the mutant strains may be related to deficiencies in the proper synthesis of cell wall components of the mycelial phase of this fungus.

Biosurfactant production by two isolates ofPseudomonas aeruginosa.

Two strains of biosurfactant-producing bacteria, identified asPseudomonas aeruginosa, were isolated from injection water and crude oil-associated water in Venezuelan oil fields. Both biosurfactants resembled rhamnolipids and produced stable emulsions of heavy and extra-heavy crude oils, reducing the surface tension of water from 72 to 28 dynes/cm. Tenso-active properties of the biosurfactants were not affected by pH, temperature, salinity or Ca(2+) or Mg(2+) at concentrations in excess of those found in many oil reservoirs in Venezuela.

Inhibition of growth of the dimorphic fungus Paracoccidioides brasiliensis by ajoene.

Ajoene, a garlic-derived compound that prevents platelet activation, inhibited the growth of Paracoccidioides brasiliensis, a fungal pathogen for humans, by affecting the integrity of the fungal cytoplasmic membrane. This action may be the basis for the study of ajoene as a possible specific antifungal drug.

Effect of papulacandin B on glucan synthesis in Paracoccidioides brasiliensis.

Papulacandin B, an inhibitor of beta-glucan synthesis in some fungi, was tested on both morphological phases of Paracoccidioides brasiliensis. In vivo, the antibiotic did not affect yeast morphology or growth but it inhibited mycelial growth and yeast----mycelium transformation. Increase of alpha-glucan and decrease of beta-glucan synthesis was also observed in the fungal cell wall of the mycelial form after addition of papulacandin B. Synthesis in vitro of beta-glucan was partially substituted by the synthesis of an amylase-sensitive glucan.

Ultrastructure of spore formation in Paracoccidioides brasiliensis.

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.

Effect of nucleotides on glucan synthesis in Paracoccidioides brasiliensis.

The activity of Paracoccidioides brasiliensis glucan synthetase was partially inhibited by guanosine 5'-triphosphate, adenosine 3'5'-phosphate and adenosine 5'-triphosphate. This inhibition was more pronounced in the mycelial system than in the yeast one, being higher at 23 degrees C than at 37 degrees C. Addition of ethene diamine tetracetic acid to the incubation mixture inhibited partially the enzymatic activity in mycelial preparations but stimulated it in the yeast system.

Chemical changes in cell wall structure of five strains of Paracoccidioides 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.