RESUMO
Aspergillus fumigatus is an important opportunistic fungal pathogen that can cause acute invasive disease in neutropenic hosts. Invasive aspergillosis is being diagnosed with increasing frequency, and morbidity and mortality remain high despite prompt antifungal therapy. Because little is known about the virulence factors used by A. fumigatus, a tissue culture model was developed to mimic the interaction of the fungus with the endothelium. Differential display was used to compare gene expression in fungal cells grown on endothelial cells with that of cells grown in the absence of endothelial cell contact, and genes that were up-regulated were selected for analysis as putatively virulence-related genes. Two of these up-regulated genes were chosen for further study and were identified as genes encoding the regulatory subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase and a member of the ras gene family, both of which are involved in cAMP-mediated signaling in fungi. This model system provides a new approach to the identification of potentially virulence-related genes induced in A. fumigatus by the interaction with host cells.
Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/genética , Endotélio Vascular/microbiologia , Regulação Fúngica da Expressão Gênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sequência de Aminoácidos , Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endotélio Vascular/citologia , Genes Fúngicos , Genes Reguladores , Genes ras , Humanos , Dados de Sequência Molecular , Veias Umbilicais , Regulação para Cima , VirulênciaAssuntos
Aspergilose/veterinária , Aspergillus/metabolismo , Doenças dos Bovinos/microbiologia , Animais , Aspergilose/microbiologia , Aspergillus/crescimento & desenvolvimento , Aspergillus/patogenicidade , Bovinos , Endopeptidases/fisiologia , Ferro/metabolismo , Metabolismo dos Lipídeos , VirulênciaRESUMO
Aspergillus flavus produces an elastinolytic metalloproteinase in culture fluid which does not appear to be a glycoprotein. The elastase did not stain with periodic acid Schiff reagent, its migration rate was not changed by digestion with glycosidases or chemical agents, and its release into culture medium was not inhibited by tunicamycin.
Assuntos
Aspergillus flavus/enzimologia , Elastase Pancreática/química , Animais , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/química , Glicosilação , Peso Molecular , Reação do Ácido Periódico de Schiff , Coelhos , Tunicamicina/farmacologiaRESUMO
An elastinolytic proteinase of Aspergillus flavus has been isolated to homogeneity, and its physical and biochemical properties have been characterized. Two purification protocols were compared; an initial step of ion-exchange chromatography was found to be equivalent to ammonium sulfate precipitation at neutral pH. A combination of gel filtration and adsorption chromatographies on the resultant crude enzyme produced highly purified elastase with yields of 5 to 10%. The enzyme is a 23-kilodalton protein with a pI of 7.6. The enzyme activity is markedly inhibited by numerous metal ions. Aspergillus elastase appears to be a metalloproteinase EC 3.4.24.X), as determined by its sensitivity to 1,10-phenanthroline.
