MOLECULAR AND CYTOLOGICAL KARYOTYPING OF Fonsecaea GENUS

Black yeasts from the Fonsecaea genus are melanized fungi responsible for a wide variety of clinical conditions, ranging from superficial, (sub) cutaneous infections, allergies and disseminated infections. Among these, chromoblastomycosis is a chronic infection that develops from a primary lesion, starting at the site of inoculation of the etiological agent and may or not advance to the subcutaneous tissues where occurs the formation of resistance cells, called muriform bodies. The low efficiency of the treatments of diseases caused by these agents makes it necessary to know better its biology. Therefore, this study aimed to establish the method of chromosome preparation and determine the karyotype of Fonsecaea genus. To realize the karyotyping two methods were combined: cytological karyotyping and Pulsed Field Gel Electrophoresis (PFGE). For cytological karyotyping 100μL of a 106 spores/mL solution was prepared, added of 200μL of thiabendazole (50mg/ml) and incubated for 3 hours, for interruption of cell division in the metaphase. Then, histological slides with those spores were stained with DAPI (4', 6-diamidino-2-phenylindole), a fluorescent dye that binds strongly in regions rich in adenine and thymine. For F. monophora electrophoretic characterization, we initially obtained protoplasts from the germinated spore solution. To that, KCl (0.7M), MgSO4 (0.4M) and NaH2PO4 (0.5M) solutions were evaluated as osmotic stabilizers. The germinated spores were solubilized in enzymatic solution with Lysing Enzymes (Sigma), followed by shaker incubation at 28°C for 3 hours. Pulsed Field Electrophoresis was performed in CHEF-DRII System (Bio-Rad) apparatus with agarose gel and 0.5x TBE buffer. After 18 hours of incubation with thiabendazole, spore germination showed higher number of viable cells for cytological karyotyping. Currently mounting of histological slides and staining with DAPI are being standardized for subsequent fluorescence microscopy. For electrophoretic karyotyping, the amount of protoplasts was higher when exposing the spores to the osmotic stabilizer MgSO4 associated to 2 hours with enzymatic solution. The agarose plugs with the protoplasts was successfully produced. To standardization the methodology of this research, the Aspergillus nidulans have being used as control organism. Furthermore, strains of albino mutants F. monophora are being produced, since the melanin present in these organisms is an effective barrier against the exit of chromosomes of protoplasts. Run conditions are being standardized