RESUMO
The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation.IMPORTANCEAspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkCA. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.
Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/enzimologia , Caspofungina/farmacologia , Parede Celular/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aspergillus fumigatus/genética , Parede Celular/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glicerol/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Concentração Osmolar , Pressão Osmótica , Fosforilação , Proteoma , Transdução de SinaisRESUMO
Here, we investigated which stress responses were influenced by the MpkC and SakA mitogen-activated protein kinases of the high-osmolarity glycerol (HOG) pathway in the fungal pathogen Aspergillus fumigatus. The ΔsakA and the double ΔmpkC ΔsakA mutants were more sensitive to osmotic and oxidative stresses, and to cell wall damaging agents. Both MpkC::GFP and SakA::GFP translocated to the nucleus upon osmotic stress and cell wall damage, with SakA::GFP showing a quicker response. The phosphorylation state of MpkA was determined post exposure to high concentrations of congo red and Sorbitol. In the wild-type strain, MpkA phosphorylation levels progressively increased in both treatments. In contrast, the ΔsakA mutant had reduced MpkA phosphorylation, and surprisingly, the double ΔmpkC ΔsakA had no detectable MpkA phosphorylation. A. fumigatus ΔsakA and ΔmpkC were virulent in mouse survival experiments, but they had a 40% reduction in fungal burden. In contrast, the ΔmpkC ΔsakA double mutant showed highly attenuated virulence, with approximately 50% mice surviving and a 75% reduction in fungal burden. We propose that both cell wall integrity (CWI) and HOG pathways collaborate, and that MpkC could act by modulating SakA activity upon exposure to several types of stresses and during CW biosynthesis.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/patogenicidade , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Biofilmes/crescimento & desenvolvimento , Parede Celular/patologia , Vermelho Congo/farmacologia , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Pressão Osmótica , Estresse Oxidativo , Fosforilação , Transdução de Sinais , Sorbitol/farmacologia , Estresse Fisiológico , VirulênciaRESUMO
Enteromorpha species are widely used as biomonitors of copper (Cu) contamination in coastal waters, but the effects of Cu at the subcellular level and possible mechanisms of metal resistance are scarcely known. To contribute to the understanding of the Cu accumulation process in macroalgae species, we exposed adult individuals of Enteromorpha flexuosa to 50, 250, and 500 microg Cu/L in seawater for 5 days under controlled conditions. Thereafter, gross photosynthesis rate (GPR) and metal accumulation were measured. Conventional transmission electron microscopy (CTEM) and energy-dispersive X-ray analysis (EDXA) were used to study the metal accumulation process at the cellular level. Treatments with 250 and 500 microg Cu/L were observed to cause an inhibition of the GPR in the algae. Cu accumulation in samples exposed to 500 microgCu/L was 5284+/-561 microg Cu/g (dry wt), whereas in control samples (no Cu addition), accumulation was 9+/-1 microg/g. In cells of plants undergoing the 50 microg Cu/L treatment, the cytoplasm was clearly vacuolated. Thickening of cell walls and increase of relative number of starch granules and of lipid bodies were the main cellular changes observed in plants exposed to 250 and 500 microg Cu/L. EDXA of algae cells after 250 and 500 microg Cu/L exposure detected Cu mainly in vacuole precipitates. Cu was also detected in chloroplasts and in some epiphytic bacteria. It was concluded that E. flexuosa did not avoid penetration of Cu into the cytoplasm and consequently its toxic effects in concentrations of 250 and 500 microg Cu/L. Precipitates containing Cu in vacuoles should be related to a metal immobilization mechanism, minimizing the Cu toxicity for cells. The epiphytic bacteria can act as a biofilter diminishing the availability of free Cu(+2) for algae accumulation.
Assuntos
Clorófitas/fisiologia , Cobre/toxicidade , Fotossíntese/efeitos dos fármacos , Poluentes da Água/toxicidade , Biodegradação Ambiental , Parede Celular/patologia , Precipitação Química , Cobre/farmacocinética , Relação Dose-Resposta a Droga , Filtração , Microscopia Eletrônica , Poluentes da Água/farmacocinéticaRESUMO
Doenca de Jorge Lobo e uma doenca cronica granulomatosa causada por um fungo obrigatorio, em cuja parede celular demonstra-se a presenca de melanina constitutiva. Ao contrario, no P. brasiliensis, e em descricoes de outros ascomicetos filogeneticamente relacionados da familia Onygenaceae, a melanina nao e detectavel pela tecnica de coloracao histoquimica de Fontana-Masson