RESUMEN
Aspergillus fumigatus is the most virulent species within the Aspergillus genus and causes invasive infections with high mortality rates. The exopolysaccharide galactosaminogalactan (GAG) contributes to the virulence of A. fumigatus. A co-regulated five-gene cluster has been identified and proposed to encode the proteins required for GAG biosynthesis. One of these genes, sph3, is predicted to encode a protein belonging to the spherulin 4 family, a protein family with no known function. Construction of an sph3-deficient mutant demonstrated that the gene is necessary for GAG production. To determine the role of Sph3 in GAG biosynthesis, we determined the structure of Aspergillus clavatus Sph3 to 1.25 Å. The structure revealed a (ß/α)8 fold, with similarities to glycoside hydrolase families 18, 27, and 84. Recombinant Sph3 displayed hydrolytic activity against both purified and cell wall-associated GAG. Structural and sequence alignments identified three conserved acidic residues, Asp-166, Glu-167, and Glu-222, that are located within the putative active site groove. In vitro and in vivo mutagenesis analysis demonstrated that all three residues are important for activity. Variants of Asp-166 yielded the greatest decrease in activity suggesting a role in catalysis. This work shows that Sph3 is a glycoside hydrolase essential for GAG production and defines a new glycoside hydrolase family, GH135.
Asunto(s)
Aspergillus fumigatus/metabolismo , Coccidioidina/química , Proteínas Fúngicas/química , Glicósido Hidrolasas/química , Polisacáridos/biosíntesis , Secuencia de Aminoácidos , Aspergillus fumigatus/enzimología , Aspergillus fumigatus/patogenicidad , Catálisis , Dominio Catalítico , Pared Celular/enzimología , Coccidioidina/genética , Coccidioidina/fisiología , Secuencia Conservada , Cristalografía por Rayos X , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/fisiología , Hidrólisis , Datos de Secuencia Molecular , Mutación , Polisacáridos/genética , Conformación Proteica , Alineación de SecuenciaRESUMEN
Aspergillus fumigatus is the most common cause of invasive mold disease in humans. The mechanisms underlying the adherence of this mold to host cells and macromolecules have remained elusive. Using mutants with different adhesive properties and comparative transcriptomics, we discovered that the gene uge3, encoding a fungal epimerase, is required for adherence through mediating the synthesis of galactosaminogalactan. Galactosaminogalactan functions as the dominant adhesin of A. fumigatus and mediates adherence to plastic, fibronectin, and epithelial cells. In addition, galactosaminogalactan suppresses host inflammatory responses in vitro and in vivo, in part through masking cell wall ß-glucans from recognition by dectin-1. Finally, galactosaminogalactan is essential for full virulence in two murine models of invasive aspergillosis. Collectively these data establish a role for galactosaminogalactan as a pivotal bifunctional virulence factor in the pathogenesis of invasive aspergillosis.
Asunto(s)
Aspergilosis/inmunología , Aspergillus fumigatus/inmunología , Aspergillus fumigatus/patogenicidad , Polisacáridos Fúngicos/inmunología , Polisacáridos/inmunología , Factores de Virulencia/inmunología , beta-Glucanos/inmunología , Animales , Aspergilosis/genética , Aspergilosis/patología , Aspergillus fumigatus/genética , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/inmunología , Línea Celular , Modelos Animales de Enfermedad , Polisacáridos Fúngicos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Humanos , Hifa/genética , Hifa/inmunología , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ratones , Polisacáridos/genética , Factores de Virulencia/genéticaRESUMEN
In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusDeltamedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The DeltamedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusDeltamedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.
