RESUMO
Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Ferro/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Metabolismo Secundário , Animais , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Cromatografia Líquida , Modelos Animais de Doenças , Deleção de Genes , Perfilação da Expressão Gênica , Aspergilose Pulmonar Invasiva/microbiologia , Aspergilose Pulmonar Invasiva/patologia , Espectrometria de Massas , Metabolômica , Camundongos , Fosfoproteínas Fosfatases/genética , Proteoma/análise , VirulênciaRESUMO
The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/metabolismo , Feminino , Proteínas Fúngicas/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos BALB C , Pressão Osmótica/fisiologia , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Esporos Fúngicos/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , VirulênciaRESUMO
Aspergillus fumigatus is a fungal pathogen that causes several invasive and noninvasive diseases named aspergillosis. This disease is generally regarded as multifactorial, considering that several pathogenicity determinants are present during the establishment of this illness. It is necessary to obtain an increased knowledge of how, and which, A. fumigatus signal transduction pathways are engaged in the regulation of these processes. Protein phosphatases are essential to several signal transduction pathways. We identified 32 phosphatase catalytic subunit-encoding genes in A. fumigatus, of which we were able to construct 24 viable deletion mutants. The role of nine phosphatase mutants in the HOG (high osmolarity glycerol response) pathway was evaluated by measuring phosphorylation of the p38 MAPK (SakA) and expression of osmo-dependent genes. We were also able to identify 11 phosphatases involved in iron assimilation, six that are related to gliotoxin resistance, and three implicated in gliotoxin production. These results present the creation of a fundamental resource for the study of signaling in A. fumigatus and its implications in the regulation of pathogenicity determinants and virulence in this important pathogen.
Assuntos
Aspergillus fumigatus/enzimologia , Proteínas Fúngicas/genética , Fosfoproteínas Fosfatases/genética , Domínio Catalítico , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Proteínas Fúngicas/classificação , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Gliotoxina/análise , Gliotoxina/metabolismo , Mutação , Fenótipo , Fosfoproteínas Fosfatases/classificação , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Filogenia , Sideróforos/análise , Transdução de Sinais , Espectrometria de Massas em Tandem , Virulência/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We have used three independent phylogenomic approaches (concatenated alignments, single-, and multi-gene supertrees) to reconstruct the fungal tree of life (FTOL) using publicly available fungal genomes. This is the first time multi-gene families have been used in fungal supertree reconstruction and permits us to use up to 66% of the 1,001,217 genes in our fungal database. Our analyses show that different phylogenomic datasets derived from varying clustering criteria and alignment orientation do not have a major effect on phylogenomic supertree reconstruction. Overall the resultant phylogenomic trees are relatively congruent with one another and successfully recover the major fungal phyla, subphyla and classes. We find that where incongruences do occur, the inferences are usually poorly supported. Within the Ascomycota phylum, our phylogenies reconstruct monophyletic Saccharomycotina and Pezizomycotina subphyla clades and infer a sister group relationship between these to the exclusion of the Taphrinomycotina. Within the Pezizomycotina subphylum, all three phylogenies infer a sister group relationship between the Leotiomycetes and Sordariomycetes classes. However, there is conflict regarding the relationships with the Dothideomycetes and Eurotiomycetes classes. Within the Basidiomycota phylum, supertrees derived from single- and multi-gene families infer a sister group relationship between the Pucciniomycotina and Agaricomycotina subphyla while the concatenated phylogeny infers a poorly supported relationship between the Agaricomycotina and Ustilagomycotina. The reconstruction of a robust FTOL is important for future fungal comparative analyses. We illustrate this point by performing a preliminary investigation into the phyletic distribution of yeast prion-like proteins in the fungal kingdom.