RESUMO
Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.
Assuntos
Proteínas da Matriz Extracelular/metabolismo , Proteínas Fúngicas/metabolismo , Paracoccidioides/química , Sequência de Aminoácidos , Animais , Feminino , Imunofluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Peso Molecular , Ligação ProteicaRESUMO
The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.
Assuntos
Anticorpos Antifúngicos/sangue , Antígenos de Fungos/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/diagnóstico , Proteínas Recombinantes/imunologia , Antígenos de Fungos/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Paracoccidioides/genética , Paracoccidioidomicose/microbiologia , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.
Assuntos
Antígenos de Fungos , Proteínas de Choque Térmico HSP70 , Paracoccidioides/imunologia , Paracoccidioidomicose/diagnóstico , Sequência de Aminoácidos , Antígenos de Fungos/análise , Antígenos de Fungos/química , Antígenos de Fungos/isolamento & purificação , Biópsia , Western Blotting , Proteínas de Choque Térmico HSP70/análise , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Choque Térmico HSP70/isolamento & purificação , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/microbiologiaRESUMO
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. Immuofluorescence 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 cell, 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 pigmented may play a role in the pathogenesis of paracoccidioidomycosis
Assuntos
Humanos , Paracoccidioides/fisiologia , Paracoccidioides/genética , Paracoccidioides/imunologia , Paracoccidioides/virologia , Pigmentação da Pele/fisiologia , Pigmentação da Pele/genéticaRESUMO
Se presentan 314 casos de micosis cutáneas en niños, que fueran diagnosticados en la Corporación para Investigaciones Biológicas (CIB), durante los años de 1987 a 1993. Se hace énfasis en la frecuencia de dermatofitosis, candidiasis y micosis superficiales. Las dermatofitosis fueron diagnosticadas en 210 pacientes (67 por ciento), con mayor frecuencia de tinea capitis y tinea corporis, 88 (42 por ciento) y 81 (38.5 por ciento) casos, respectivamente. La tinea pedis también ocupó lugar importante con 27 casos (13 por ciento). Entre los agentes etiológicos, Microsporum canis fue aislado de 94 pacientes con predominio de las lesiones de cuero cabelludo, 75 casos (80 por ciento). El M.gypseum ocupó el segundo lugar con 52 casos (25 por ciento) y fué además el principal causante de tinea corporis (20.7 por ciento) en este grupo de pacientes. La candidiasis se diagnosticó en 89 pacientes (28 por ciento); la localización más común fue la de la piel glabra (35 por ciento). Candida albicans fue la especie aislada con mayor frecuencia (50.5 por ciento), seguido por C.parapsilosis, 19 casos (21 por ciento). Entre las micosis superficiales, la pitiriasis versicolor se observó en 14 casos (4 por ciento), mientras que la piedra negra fue encontrada sólo en una oportunidad. Los resultados anotados permiten señalar la importancia de las micosis dérmicas en la población pediátrica