RESUMEN
While most Ascomycetes tend to associate principally with plants, the dimorphic fungi Coccidioides immitis and Coccidioides posadasii are primary pathogens of immunocompetent mammals, including humans. Infection results from environmental exposure to Coccidiodies, which is believed to grow as a soil saprophyte in arid deserts. To investigate hypotheses about the life history and evolution of Coccidioides, the genomes of several Onygenales, including C. immitis and C. posadasii; a close, nonpathogenic relative, Uncinocarpus reesii; and a more diverged pathogenic fungus, Histoplasma capsulatum, were sequenced and compared with those of 13 more distantly related Ascomycetes. This analysis identified increases and decreases in gene family size associated with a host/substrate shift from plants to animals in the Onygenales. In addition, comparison among Onygenales genomes revealed evolutionary changes in Coccidioides that may underlie its infectious phenotype, the identification of which may facilitate improved treatment and prevention of coccidioidomycosis. Overall, the results suggest that Coccidioides species are not soil saprophytes, but that they have evolved to remain associated with their dead animal hosts in soil, and that Coccidioides metabolism genes, membrane-related proteins, and putatively antigenic compounds have evolved in response to interaction with an animal host.
Asunto(s)
Coccidioides/genética , Genoma Fúngico , Hongos Mitospóricos/genética , Animales , Especiación Genética , Genómica/métodos , Histoplasma/genética , Humanos , Datos de Secuencia Molecular , Onygenales/genética , Filogenia , Selección Genética , Análisis de Secuencia de ADN , SinteníaRESUMEN
BACKGROUND: Tetraspanins are small membrane proteins that belong to a superfamily encompassing 33 members in human and mouse. These proteins act as organizers of membrane-signalling complexes. So far only two tetraspanin families have been identified in fungi. These are Pls1, which is required for pathogenicity of the plant pathogenic ascomycetes, Magnaporthe grisea, Botrytis cinerea and Colletotrichum lindemuthianum, and Tsp2, whose function is unknown. In this report, we describe a third family of tetraspanins (Tsp3) and a new family of tetraspanin-like proteins (Tpl1) in fungi. We also describe expression of some of these genes in M. grisea and a basidiomycete, Laccaria bicolor, and also their functional analysis in M. grisea. RESULTS: The exhaustive search for tetraspanins in fungal genomes reveals that higher fungi (basidiomycetes and ascomycetes) contain three families of tetraspanins (Pls1, Tsp2 and Tsp3) with different distribution amongst phyla. Pls1 is found in ascomycetes and basidiomycetes, whereas Tsp2 is restricted to basidiomycetes and Tsp3 to ascomycetes. A unique copy of each of PLS1 and TSP3 was found in ascomycetes in contrast to TSP2, which has several paralogs in the basidiomycetes, Coprinus cinereus and Laccaria bicolor. A tetraspanin-like family (Tpl1) was also identified in ascomycetes. Transcriptional analyses in various tissues of L. bicolor and M. grisea showed that PLS1 and TSP2 are expressed in all tissues in L. bicolor and that TSP3 and TPL1 are overexpressed in the sexual fruiting bodies (perithecia) and mycelia of M. grisea, suggesting that these genes are not pseudogenes. Phenotypic analysis of gene replacementmutants Deltatsp3 and Deltatpl1 of M. grisea revealed a reduction of the pathogenicity only on rice, in contrast to Deltapls1 mutants, which are completely non-pathogenic on barley and rice. CONCLUSION: A new tetraspanin family (Tsp3) and a tetraspanin-like protein family (Tpl1) have been identified in fungi. Functional analysis by gene replacement showed that these proteins, as well as Pls1, are involved in the infection process of the plant pathogenic fungus M. grisea. The next challenge will be to decipher the role(s) of tetraspanins in a range of symbiotic, saprophytic and human pathogenic fungi.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Hongos/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Hongos/fisiología , Genoma Fúngico , Magnaporthe/genética , Magnaporthe/fisiología , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Alineación de SecuenciaRESUMEN
Coccidioidomycosis is a mild to life-threatening disease in otherwise healthy humans and other mammals caused by the fungus Coccidioides spp. Understanding the development of the unique dimorphic life cycle of Coccidioides spp. and its role in pathogenesis has been an area of research focus. However, nuclear behavior during the saprobic and parasitic life cycle has not been studied intensively. In this study, green fluorescent protein (GFP) was fused to histone H1 and introduced into Coccidioides posadasii (C. posadasii) strain Silveira to monitor the nuclear behavior of the fungus during the saprobic and parasitic stages of the life cycle. We constructed an Agrobacterium tumefaciens-mediated transformation (ATMT) vector that had in its T-DNA region a hygromycin-resistance gene as well as the fused histone H1-GFP gene under the control of the histone H3 promoter of C. posadasii. More than 30 hygromycin-resistant transformants were obtained and 23 were purified to homozygosity through multiple passages of the original transformants on hygromycin-containing media. One strain (VFC1420) transformed with a single copy of the fusion histone H1-GFP gene was selected for cytological studies. Strong nuclear-localized GFP signals were observed in arthroconidia, hyphae, as well as in spherules and endospores developed in vitro. Thus GFP can be used to study the expression pattern of potential virulence genes identified in serial analysis of gene expression (SAGE) or expressed sequence tags (EST) libraries, and could be a useful tool to monitor disease development in the murine model.
Asunto(s)
Núcleo Celular/metabolismo , Coccidioides/metabolismo , Coccidioidomicosis/genética , Proteínas Fluorescentes Verdes/metabolismo , Microscopía Fluorescente/instrumentación , Agrobacterium tumefaciens/metabolismo , Coccidioidomicosis/metabolismo , Etiquetas de Secuencia Expresada , Técnicas Genéticas , Vectores Genéticos , Humanos , Microscopía Fluorescente/métodos , Regiones Promotoras GenéticasRESUMEN
Conidiophore morphogenesis in Aspergillus nidulans occurs in response to developmental signals that result in the activation of brlA, a well-characterized gene that encodes a transcription factor that is central to asexual development. Loss-of-function mutations in flbD and other fluffy loci have previously been shown to result in delayed development and reduced expression of brlA. flbD message is detectable during both hyphal growth and conidiation, and its gene product is similar to the Myb family of transcription factors. To further understand the regulatory pathway to brlA activation and conidiation, we isolated suppressor mutations that rescued development in strains with a flbD null allele. We describe here two new loci, designated sfdA and sfdB for suppressors of flbD, that bypass the requirement of flbD for development. sfd mutant alleles were found to restore developmental timing and brlA expression to strains with flbD deletions. In addition, sfd mutations suppress the developmental defects in strains harboring loss-of-function mutations in fluG, flbA, flbB, flbC, and flbE. All alleles of sfdA and sfdB that we have isolated are recessive to their wild-type alleles in diploids. Strains with mutant sfd alleles in otherwise developmentally wild-type backgrounds have reduced growth phenotypes and develop conidiophores in submerged cultures.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Fúngica de la Expresión Génica/fisiología , Genes Fúngicos , Supresión Genética , Factores de Transcripción , Aspergillus nidulans/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/fisiología , Ligamiento Genético , Transactivadores/genética , Transactivadores/fisiologíaRESUMEN
Coccidioides posadasii and Coccidioides immitis are dimorphic, soil-dwelling pathogenic ascomycetes endemic to the southwestern United States. Infection can result from inhalation of a very few arthroconidia, but following natural infection, long-lived immunity is the norm. Previous work in the field has shown that spherule-derived vaccines afford more protection than those from mycelia. We have used two-dimensional differential in-gel electrophoresis coupled with nano-high-performance liquid chromatography-tandem mass spectrometry to directly assess both absolute abundance and differential expression of proteins in the spherule and the mycelial phases of C. posadasii with the intent to identify potential vaccine candidates. Peptides derived from 40 protein spots were analyzed and a probable identity was assigned to each. One spherule-abundant protein, identified as Pmp1, showed homology to allergens from Aspergillus fumigatus and other fungi, all of which exhibit similarity to yeast thiol peroxidases. Recombinant Pmp1 was reactive with serum from individuals with both acute and protracted disease, and evoked protection in two murine models of infection with C. posadasii. These results demonstrate the utility of proteomic analysis as a point of discovery for protective antigens for possible inclusion in a vaccine candidate to prevent coccidioidomycosis.
Asunto(s)
Coccidioides , Coccidioidomicosis/metabolismo , Proteínas Fúngicas/análisis , Vacunas Fúngicas/administración & dosificación , Peroxisomas/química , Animales , Coccidioidomicosis/prevención & control , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Vacunas Fúngicas/inmunología , Proteínas de Transporte de Membrana/inmunología , Proteínas de Transporte de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas/instrumentación , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
1,3-beta-Glucan synthase is responsible for the synthesis of beta-glucan, an essential cell wall structural component in most fungi. We sought to determine whether Coccidioides posadasii possesses genes homologous to known fungal FKS genes that encode the catalytic subunit of 1,3-beta-glucan synthase. A single gene, designated FKS1, was identified, and examination of its predicted protein product showed a high degree of conservation with Fks proteins from other filamentous fungi. FKS1 is expressed at similar levels in mycelia and early spherulating cultures, and expression decreases as the spherules mature. We used Agrobacterium-mediated transformation to create strains that harbor DeltaFKS1::hygB, a null allele of FKS1, and hypothesize that Fks1p function is essential, due to our inability to purify this allele away from a complementing wild-type FKS1 allele in a heterokaryotic strain. The heterokaryon appears normal with respect to growth rate and arthroconidium production; however, microscopic examination of strains with DeltaFKS1::hygB alleles revealed abnormal swelling of hyphal elements.