RESUMO
Paracoccidioidomycosis is an important systemic mycosis caused by thermodimorphic fungi of the Paracoccidioides genus. During the infective process, the cell wall acts at the interface between the fungus and the host. In this way, the cell wall has a key role in growth, environment sensing and interaction, as well as morphogenesis of the fungus. Since the cell wall is absent in mammals, it may present molecules that are described as target sites for new antifungal drugs. Despite its importance, up to now few studies have been conducted employing proteomics in for the identification of cell wall proteins in Paracoccidioides spp. Here, a detailed proteomic approach, including cell wall-fractionation coupled to NanoUPLC-MSE, was used to study and compare the cell wall fractions from Paracoccidioides lutzii mycelia and yeast cells. The analyzed samples consisted of cell wall proteins extracted by hot SDS followed by extraction by mild alkali. In summary, 512 proteins constituting different cell wall fractions were identified, including 7 predicted GPI-dependent cell wall proteins that are potentially involved in cell wall metabolism. Adhesins previously described in Paracoccidioides spp. such as enolase, glyceraldehyde-3-phosphate dehydrogenase were identified. Comparing the proteins in mycelium and yeast cells, we detected some that are common to both fungal phases, such as Ecm33, and some specific proteins, as glucanase Crf1. All of those proteins were described in the metabolism of cell wall. Our study provides an important elucidation of cell wall composition of fractions in Paracoccidioides, opening a way to understand the fungus cell wall architecture.
Assuntos
Parede Celular/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Micélio/genética , Paracoccidioides/genética , Bicarbonatos/química , Parede Celular/química , Cromatografia Líquida , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Ontologia Genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/isolamento & purificação , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Extração Líquido-Líquido/métodos , Redes e Vias Metabólicas/genética , Anotação de Sequência Molecular , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioides/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/isolamento & purificação , Fosfopiruvato Hidratase/metabolismo , Proteômica/métodos , Dodecilsulfato de Sódio/química , Espectrometria de Massas em TandemRESUMO
Black fungi comprise a diverse group of melanized microorganisms, many of which are able to infect humans. One of the recognized diseases that arise with black fungi infection is chromoblastomycosis, a neglected implantation mycosis. Considering their ecology, black fungi may face conditions with distinct metal availability. Zinc and copper are essential transition metals, which become toxic in excess. During the interaction with host, fungi may face either metal deprivation or poisoning. Here we report an in silico analysis of four black fungi genomes concerning zinc and copper homeostasis. Overall, these organisms share apparatus of metal uptake, storage and detoxification with other pathogenic and non-pathogenic fungi. Genes coding plasma membrane and organelle transporters, as well as metal binding proteins were identified. Althought putatives zinc and copper responsive transcription factors have been found in the analyzed genomes, remarkable structural differences were perceived when compared to the already characterized regulators. Black fungi may harbor unique features concerning the regulation of zinc and copper homeostasis, which is probably a result of the niches they can inhabit. The data provided here add knowlegde to a still unexplored aspect of black fungi biology that may be useful in the understanding of their pathogenicity.
Assuntos
Ascomicetos/metabolismo , Cromoblastomicose/microbiologia , Cobre/metabolismo , Zinco/metabolismo , Ascomicetos/classificação , Ascomicetos/genética , Ascomicetos/patogenicidade , Homeostase , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MSE. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to ß-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.