RESUMEN
Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.
Asunto(s)
Melaninas/metabolismo , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/microbiología , Animales , Medios de Cultivo , Espectroscopía de Resonancia por Spin del Electrón , Lacasa , Levodopa/metabolismo , Pulmón/metabolismo , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Oxidorreductasas/metabolismo , Paracoccidioides/metabolismo , Bazo/metabolismo , Bazo/microbiología , VirulenciaRESUMEN
Phenotypic variability in pathogenic fungi has long been correlated with virulence, but specific genetic and molecular mechanisms are only recently being unraveled. Fungal morphogenesis, reflecting the expression of several regulated genes, and the capacity of the rising forms or phases to cause disease has been focused on at the XIVth Congress of the International Society for Human and Animal Mycology. Three experimental models of pathogenic fungi have been discussed. In Cryptococcus neoformans, phenotypic variability or switching represents controlled and programmed changes rather than random mutations. Evaluated phenotypic traits were the capsular polysaccharide, cell and colony morphology and virulence. In the dimorphic Paracoccidioides brasiliensis, the serine-thiol proteinase from the yeast phase cleaves the main components of the basal membrane, thus being potentially relevant in fungal dissemination. In Candida albicans, relationships between adhesion proteins and those of lymphocytes and neutrophils are related to fungal pathogenicity. Regulation of the directional growth of hyphae and its tropic responses are correlated with the invasive potential of C. albicans.
Asunto(s)
Candida albicans/patogenicidad , Cryptococcus neoformans/patogenicidad , Paracoccidioides/patogenicidad , Candida albicans/genética , Candida albicans/crecimiento & desarrollo , Secuencia de Carbohidratos , Cryptococcus neoformans/genética , Cryptococcus neoformans/crecimiento & desarrollo , Humanos , Datos de Secuencia Molecular , Morfogénesis , Micosis/microbiología , Paracoccidioides/genética , Paracoccidioides/crecimiento & desarrollo , VirulenciaRESUMEN
The deoxynucleotide (dNMP) composition of ten strains of C. neoformans was analysed by 32P-labelling and two-dimensional thin-layer chromatography. This technique is very sensitive for detecting rare deoxynucleotide adducts and analogues (minor bases) in DNA. The results indicate considerable variation among strains in DNA nucleotide composition.
Asunto(s)
Cryptococcus neoformans/genética , ADN de Hongos/química , Desoxirribonucleótidos/análisis , Variación Genética , Autorradiografía , Composición de Base , Brasil , Cromatografía en Capa Delgada/métodos , Cryptococcus neoformans/aislamiento & purificación , ADN de Hongos/genética , ADN de Hongos/aislamiento & purificación , Meningitis Criptocócica/líquido cefalorraquídeo , Meningitis Criptocócica/microbiología , Reproducibilidad de los Resultados , Especificidad de la EspecieRESUMEN
Little is known about the global molecular epidemiology of the human pathogenic fungus Cryptococcus neoformans. We studied 51 clinical and environmental (pigeon excreta) isolates from two cities in Brazil (Belo Horizonte and Rio de Janeiro) by analyzing their carbon assimilation patterns, electrophoretic karyotypes, restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element-1 (CNRE-1), and URA5 sequences. Results were compared to those previously obtained for isolates from New York City by the same DNA typing methods. Computer-assisted analysis of RFLPs and contour-clamped homogeneous electrophoresis (CHEF) patterns and URA5 sequences was performed to generate dendrograms. Some environmental and clinical isolates were found to be indistinguishable by CHEF, CNRE-1 RFLP, and URA5 sequence analyses. Similarly, some isolates from Rio de Janeiro and Belo Horizonte were indistinguishable by the three DNA typing techniques. Overall, Brazilian isolates appeared to be less heterogeneous by DNA analysis than isolates from other regions. Several Brazilian isolates were highly related to New York City isolates. Phylogenetic analysis of the sequences obtained for the Brazilian isolates and those obtained for New York City isolates was congruent with the dendrogram generated from the CNRE-1 RFLP data. In summary our results indicate (i) that the discriminatory power of the DNA typing method differs for Brazilian and New York City strains, with the order being CNRE-1 RFLP analysis > URA5 sequence analysis > CHEF analysis and CHEF analysis > URA5 sequence analysis > CNRE-1 RFLP analysis, respectively; (ii) that there are differences in local genetic diversity for Brazilian and New York City isolates; (iii) that there is additional evidence linking clinical isolates to those in pigeon excreta; and (iv) that some isolates from Brazil and New York City are closely related, consistent with the global dispersal of certain pathogenic strains.