RESUMO
Aspergillus fumigatus is a saprophytic fungus that can cause a variety of human diseases known as aspergillosis. Mycotoxin gliotoxin (GT) production is important for its virulence and must be tightly regulated to avoid excess production and toxicity to the fungus. GT self-protection by GliT oxidoreductase and GtmA methyltransferase activities is related to the subcellular localization of these enzymes and how GT can be sequestered from the cytoplasm to avoid increased cell damage. Here, we show that GliT:GFP and GtmA:GFP are localized in the cytoplasm and in vacuoles during GT production. The Mitogen-Activated Protein kinase MpkA is essential for GT production and self-protection, interacts physically with GliT and GtmA and it is necessary for their regulation and subsequent presence in the vacuoles. The sensor histidine kinase SlnASln1 is important for modulation of MpkA phosphorylation. Our work emphasizes the importance of MpkA and compartmentalization of cellular events for GT production and self-defense.
Assuntos
Aspergilose , Gliotoxina , Humanos , Aspergillus fumigatus/metabolismo , Gliotoxina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aspergilose/microbiologiaRESUMO
Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.
Assuntos
Celulose , Saccharum , Aspergillus fumigatus/metabolismo , Oxigenases de Função Mista , Saccharum/metabolismo , Saccharum/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , PolissacarídeosRESUMO
Thermotolerance is a remarkable virulence attribute of Aspergillus fumigatus, but the consequences of heat shock (HS) to the cell membrane of this fungus are unknown, although this structure is one of the first to detect changes in ambient temperature that imposes on the cell a prompt adaptative response. Under high-temperature stress, fungi trigger the HS response controlled by heat shock transcription factors, such as HsfA, which regulates the expression of heat shock proteins. In yeast, smaller amounts of phospholipids with unsaturated fatty acid (FA) chains are synthesized in response to HS, directly affecting plasma membrane composition. The addition of double bonds in saturated FA is catalyzed by Δ9-fatty acid desaturases, whose expression is temperature-modulated. However, the relationship between HS and saturated/unsaturated FA balance in membrane lipids of A. fumigatus in response to HS has not been investigated. Here, we found that HsfA responds to plasma membrane stress and has a role in sphingolipid and phospholipid unsaturated biosynthesis. In addition, we studied the A. fumigatus Δ9-fatty acid desaturase sdeA and discovered that this gene is essential and required for unsaturated FA biosynthesis, although it did not directly affect the total levels of phospholipids and sphingolipids. sdeA depletion significantly sensitizes mature A. fumigatus biofilms to caspofungin. Also, we demonstrate that hsfA controls sdeA expression, while SdeA and Hsp90 physically interact. Our results suggest that HsfA is required for the adaptation of the fungal plasma membrane to HS and point out a sharp relationship between thermotolerance and FA metabolism in A. fumigatus. IMPORTANCE Aspergillus fumigatus causes invasive pulmonary aspergillosis, a life-threatening infection accounting for high mortality rates in immunocompromised patients. The ability of this organism to grow at elevated temperatures is long recognized as an essential attribute for this mold to cause disease. A. fumigatus responds to heat stress by activating heat shock transcription factors and chaperones to orchestrate cellular responses that protect the fungus against damage caused by heat. Concomitantly, the cell membrane must adapt to heat and maintain physical and chemical properties such as the balance between saturated/unsaturated fatty acids. However, how A. fumigatus connects these two physiological responses is unclear. Here, we explain that HsfA affects the synthesis of complex membrane lipids such as phospholipids and sphingolipids and controls the enzyme SdeA, which produces monounsaturated fatty acids, raw material for membrane lipids. These findings suggest that forced dysregulation of saturated/unsaturated fatty acid balance might represent novel strategies for antifungal therapy.
Assuntos
Aspergillus fumigatus , Termotolerância , Humanos , Aspergillus fumigatus/genética , Aspergillus fumigatus/metabolismo , Termotolerância/fisiologia , Fatores de Transcrição de Choque Térmico/metabolismo , Ácidos Graxos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fosfolipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Esfingolipídeos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Concern over environmental impacts has spurred many efforts to replace fossil fuels with biofuels such as ethanol. However, for this to be possible, it is necessary to invest in other production technologies, such as second generation (2G) ethanol, in order to raise the levels of this product and meet the growing demand. Currently, this type of production is not yet economically feasible, due to the high costs of the enzyme cocktails used in saccharification stage of lignocellulosic biomass. In order to optimize these cocktails, the search for enzymes with superior activities has been the goal of several research groups. For this end, we have characterized the new ß-glycosidase AfBgl1.3 from A. fumigatus after expression and purification in Pichia pastoris X-33. Structural analysis by circular dichroism revealed that increasing temperature destructured the enzyme; the apparent Tm value was 48.5 °C. The percentages of α-helix (36.3%) and ß-sheet (12.4%) secondary structures at 25 °C were predicted. Biochemical characterization suggested that the optimal conditions for AfBgl1.3 were pH 6.0 and temperature of 40 °C. At 30 and 40 °C, the enzyme was stable and retained about 90% and 50% of its activity, respectively, after pre-incubation for 24 h. In addition, the enzyme was highly stable at pH between 5 and 8, retaining over 65% of its activity after pre-incubation for 48 h. AfBgl1.3 co-stimulation with 50-250 mM glucose enhanced its specific activity by 1.4-fold and revealed its high tolerance to glucose (IC50 = 2042 mM). The enzyme was active toward the substrates salicin (495.0 ± 49.0 U mg-1), pNPG (340.5 ± 18.6 U mg-1), cellobiose (89.3 ± 5.1 U mg-1), and lactose (45.1 ± 0.5 U mg-1), so it had broad specificity. The Vmax values were 656.0 ± 17.5, 706.5 ± 23.8, and 132.6 ± 7.1 U mg-1 toward p-nitrophenyl-ß-D-glucopyranoside (pNPG), D-(-)-salicin, and cellobiose, respectively. AfBgl1.3 displayed transglycosylation activity, forming cellotriose from cellobiose. The addition of AfBgl1.3 as a supplement at 0.9 FPU/g of cocktail Celluclast® 1.5L increased carboxymethyl cellulose (CMC) conversion to reducing sugars (g L-1) by about 26% after 12 h. Moreover, AfBgl1.3 acted synergistically with other Aspergillus fumigatus cellulases already characterized by our research group-CMC and sugarcane delignified bagasse were degraded, releasing more reducing sugars compared to the control. These results are important in the search for new cellulases and in the optimization of enzyme cocktails for saccharification.
Assuntos
Aspergillus fumigatus , Glicosídeo Hidrolases , Aspergillus fumigatus/metabolismo , Glicosídeo Hidrolases/metabolismo , Celobiose , Glucose/metabolismo , beta-Glucosidase/metabolismo , Etanol/metabolismo , Concentração de Íons de Hidrogênio , HidróliseRESUMO
Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe3+ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MSE. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Aspergillus fumigatus/metabolismo , Ferro/metabolismo , Saccharomyces cerevisiae/metabolismo , Sideróforos/metabolismoRESUMO
Aspergillus fumigatus is a major opportunistic fungal pathogen of immunocompromised and immunocompetent hosts. To successfully establish an infection, A. fumigatus needs to use host carbon sources, such as acetate, present in the body fluids and peripheral tissues. However, utilization of acetate as a carbon source by fungi in the context of infection has not been investigated. This work shows that acetate is metabolized via different pathways in A. fumigatus and that acetate utilization is under the regulatory control of a transcription factor (TF), FacB. A. fumigatus acetate utilization is subject to carbon catabolite repression (CCR), although this is only partially dependent on the TF and main regulator of CCR CreA. The available extracellular carbon source, in this case glucose and acetate, significantly affected A. fumigatus virulence traits such as secondary metabolite secretion and cell wall composition, with the latter having consequences for resistance to oxidative stress, antifungal drugs, and human neutrophil-mediated killing. Furthermore, deletion of facB significantly impaired the in vivo virulence of A. fumigatus in both insect and mammalian models of invasive aspergillosis. This is the first report on acetate utilization in A. fumigatus, and this work further highlights the importance of available host-specific carbon sources in shaping fungal virulence traits and subsequent disease outcome, and a potential target for the development of antifungal strategies. IMPORTANCE Aspergillus fumigatus is an opportunistic fungal pathogen in humans. During infection, A. fumigatus is predicted to use host carbon sources, such as acetate, present in body fluids and peripheral tissues, to sustain growth and promote colonization and invasion. This work shows that A. fumigatus metabolizes acetate via different pathways, a process that is dependent on the transcription factor FacB. Furthermore, the type and concentration of the extracellular available carbon source were determined to shape A. fumigatus virulence determinants such as secondary metabolite secretion and cell wall composition. Subsequently, interactions with immune cells are altered in a carbon source-specific manner. FacB is required for A. fumigatus in vivo virulence in both insect and mammalian models of invasive aspergillosis. This is the first report that characterizes acetate utilization in A. fumigatus and highlights the importance of available host-specific carbon sources in shaping virulence traits and potentially subsequent disease outcome.
Assuntos
Acetatos/metabolismo , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/genética , Proteínas Fúngicas/metabolismo , Humanos , Larva/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Neutrófilos/microbiologia , Fenótipo , Metabolismo Secundário , VirulênciaRESUMO
The recovery of metals from electronic waste was investigated by using fungal strain Aspergillus fumigatus A2DS, isolated from the mining industry wastewater. Fifty-seven percent of copper and 32% of nickel were leached (analyzed by Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-OES)) by the organism after one-step leaching at a temperature of 30 °C (shaking condition for 7 days). Maximum % of copper and nickel were obtained at a pH of 6 (58.7% Cu and 32% Ni), the temperature of 40 °C (61.8% Cu and 27.07% Ni), a pulp density of 0.5% (62% Cu and 42.37% Ni), and inoculums of 1% (58% Cu and 32.29% Ni). The XRD pattern of PCB showed 77.6% of copper containing compounds. XRD analysis of the leachate residue showed only 23.2% Euchorite (ASCu2H7O8) and 9.4% other copper containing compounds, indicating the leaching property of the fungus. HPLC analysis of the spent medium showed the presence of different acids like citric, succinic, and fumaric acid. The FTIR spectrum showed a decrease in carboxylic stretching in the leachate produced after bioleaching using spent medium. ICPOES of the leachate obtained using spent medium showed that 61% of the copper and 35% of nickel were leached out after seven days of incubation at shaking condition and 57% of copper and 32.8% of nickel at static condition confirming acidolysis property of the strain. A. fumigatus A2DS metal absorption and adsorption ability were observed using transmission electron microscopy (TEM) and scanning electron microscopy energy dispersive X-ray (SEM-EDX) respectively. The results thus indicate that bioleaching of Cu and Ni is bioleached by A. fumigatus A2DS.
Assuntos
Aspergillus fumigatus/metabolismo , Telefone Celular , Cobre/isolamento & purificação , Resíduo Eletrônico , Níquel/isolamento & purificação , MetaisRESUMO
The main objective of the study is to characterize two new strains of Aspergillus fumigatus through morphometric, biochemical, molecular methods, and to evaluate their antimicrobial potentiality. The micro-morphotaxonomy, growth, and metabolic behavior of the strains, nHF-01 and PPR-01, were studied in different growth conditions and compared with standard strain. The molecular characterization was done by sequencing the ncrDNA ITS1-5.8S-ITS2 and D1-D2 domains of the nc 28S rDNA region and compared with a secondary structure-based phylogenetic tree. The secretory antimicrobials and pigments were characterized by TLC, UV-Vis, and FT-IR spectroscopy. Both the strains showed distinct growth patterns in different nutritional media and could assimilate a wide range of carbohydrates with distinctive biochemical properties. The molecular characterization revealed the strains, nHF-01 and PPR-01, as Aspergillus fumigatus (GenBank Accession No. MN190286 and MN190284, respectively). It was observed that the strain nHF-01 produces red to brownish pigments having mild antimicrobial activity while the strain PPR-01 does not represent such transformations. The extractable compounds had a significant antimicrobial potentiality against the human pathogenic bacteria. From this analysis, it can be concluded that the nHF-01 and PPR-01 strains are distinct from other A. fumigatus by their unique characters. Large-scale production and detailed molecular elucidation of the antimicrobial compounds may lead to the discovery of new antimicrobial compounds from these strains.
Assuntos
Anti-Infecciosos/metabolismo , Aspergillus fumigatus/metabolismo , Anti-Infecciosos/farmacologia , Aspergillus fumigatus/classificação , Aspergillus fumigatus/genética , Aspergillus fumigatus/crescimento & desenvolvimento , Meios de Cultura/química , DNA Fúngico/genética , DNA Ribossômico/genética , Humanos , Filogenia , Pigmentos Biológicos/metabolismo , Pigmentos Biológicos/farmacologia , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
Aspergillus fumigatus produces diverse secondary metabolites whose biological functions and regulation remain to be understood. Despite the importance of the conidia for this fungus, the role of the conidia-born metabolite fumiquinazoline C (FqC) is unclear. Here, we describe a dual function of the cell-wall integrity pathway in regulating FqC biosynthesis dictated by the MAPK kinase MpkA, which phosphorylates one of the nonribosomal peptide synthetases enzymes of the cluster (FmqC), and the transcription factor RlmA, which directly regulates the expression of fmq genes. Another level of crosstalk between the FqC regulation and the cell physiology is described since the deletion of the stress-responsive transcription factor sebA provokes derepression of the fmq cluster and overproduction of FqC. Thus, we describe a mechanism by which A. fumigatus controls FqC biosynthesis orchestrated by MpkA-RlmA and SebA and hence enabling survival and adaptation to the environmental niche, given that FqC is a deterrent of ameba predation.
Assuntos
Aspergillus fumigatus/genética , Quinazolinas/metabolismo , Aspergillus fumigatus/metabolismo , Parede Celular/genética , Proteínas Fúngicas/genética , Expressão Gênica , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fagocitose/fisiologia , Transdução de Sinais , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismo , Transcrição GênicaRESUMO
The fungal zinc finger transcription factor NsdC is named after, and is best known for, its essential role in sexual reproduction (never in sexual development). In previous studies with Aspergillus nidulans, it was also shown to have roles in promotion of vegetative growth and suppression of asexual conidiation. In this study, the function of the nsdC homologue in the opportunistic human pathogen A. fumigatus was investigated. NsdC was again found to be essential for sexual development, with deletion of the nsdC gene in both MAT1-1 and MAT1-2 mating partners of a cross leading to complete loss of fertility. However, a functional copy of nsdC in one mating partner was sufficient to allow sexual reproduction. Deletion of nsdC also led to decreased vegetative growth and allowed conidiation in liquid cultures, again consistent with previous findings. However, NsdC in A. fumigatus was shown to have additional biological functions including response to calcium stress, correct organization of cell wall structure, and response to the cell wall stressors. Furthermore, virulence and host immune recognition were affected. Gene expression studies involving chromatin immunoprecipitation (ChIP) of RNA polymerase II (PolII) coupled to next-generation sequencing (Seq) revealed that deletion of nsdC resulted in changes in expression of over 620 genes under basal growth conditions. This demonstrated that this transcription factor mediates the activity of a wide variety of signaling and metabolic pathways and indicates that despite the naming of the gene, the promotion of sexual reproduction is just one among multiple roles of NsdC.IMPORTANCEAspergillus fumigatus is an opportunistic human fungal pathogen and the main causal agent of invasive aspergillosis, a life-threatening infection especially in immunocompromised patients. A. fumigatus can undergo both asexual and sexual reproductive cycles, and the regulation of both cycles involves several genes and pathways. Here, we have characterized one of these genetic determinants, the NsdC transcription factor, which was initially identified in a screen of transcription factor null mutants showing sensitivity when exposed to high concentrations of calcium. In addition to its known essential roles in sexual reproduction and control of growth rate and asexual reproduction, we have shown in the present study that A. fumigatus NsdC transcription factor has additional previously unrecognized biological functions including calcium tolerance, cell wall stress response, and correct cell wall organization and functions in virulence and host immune recognition. Our results indicate that NsdC can play novel additional biological functions not directly related to its role played during sexual and asexual processes.