RESUMEN
Aromatic compounds serve as the primary source of floral and fruity aromas in sauce-flavor (Maotai flavor) baijiu, constituting the skeleton components of its flavor profile. Nevertheless, the formation mechanism of these compounds and key aroma-producing enzymes in sauce-flavor Daqu (fermentation agent, SFD) remain elusive. Here, we combined metagenomics, metaproteomics, metabolomics, and key enzyme activity to verify the biosynthesis pathway of aromatic compounds and to identify key enzymes, genes, and characteristic microorganisms in SFD. The results showed that the later period of fermentation was critical for the generation of aromatic compounds in SFD. In-situ verification was conducted on the potential key enzymes and profiles in various metabolites, providing comprehensive evidence for the main synthetic pathways of aromatic compounds in SFD. Notably, our results showed that primary amine oxidase (PrAO) and aldehyde dehydrogenase (ALDH) emerged as two key enzymes promoting aromatic compound synthesis. Additionally, two potential key functional genes regulating aromatics generation were identified during SFD fermentation through correlation analysis between proteins and relevant metabolites, coupled with in vitro amplification test. Furthermore, original functional strains (Aspergillus flavus-C10 and Aspergillus niger-IN2) exhibiting high PrAO and ALDH production were successfully isolated from SFD, thus validating the results of metagenomics and metaproteomics analyses. This study comprehensively elucidates the pathway of aromatic compound formation in SFD at the genetic, proteomic, enzymatic, and metabolomic levels, providing new ideas for the investigation of key flavor substances in baijiu. Additionally, these findings offer valuable insights into the regulatory mechanisms of aromatic compounds generation.
Asunto(s)
Fermentación , Aromatizantes , Aromatizantes/metabolismo , Odorantes/análisis , Proteómica , Aspergillus niger/enzimología , Aspergillus niger/genética , Aspergillus niger/metabolismo , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Metagenómica , Metabolómica , Alimentos Fermentados/microbiologíaRESUMEN
The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.
Asunto(s)
Aflatoxinas , Amidohidrolasas , Aspergillus flavus , Aspergillus flavus/genética , Aspergillus flavus/enzimología , Aspergillus flavus/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Aflatoxinas/biosíntesis , Aflatoxinas/metabolismo , Aflatoxinas/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Quitina/metabolismo , Pared Celular/metabolismoRESUMEN
The Fus3-MAP kinase module is a conserved phosphorylation signal system in eukaryotes that responds to environmental stress and transduction of external signals from the outer membrane to the nucleus. Aspergillus flavus can produce aflatoxins (AF), which seriously threaten human and animal health. In this study, we determined the functions of Fus3, confirmed Ste50-Ste11-Ste7-Fus3 protein interactions and phosphorylation, and explored the possible phosphorylation motifs and potential targets of Fus3. The regulatory mechanism of Fus3 on the biosynthesis of AF was partly revealed in this study. AF production was downregulated in Δfus3, but the transcriptional expression of most AF cluster genes was upregulated. It is notable that the levels of acetyl-CoA and malonyl-CoA, the substrates of AF, were significantly decreased in fus3 defective strains. Genes involved in acetyl-CoA and malonyl-CoA biosynthesis were significantly downregulated at transcriptional or phosphorylation levels. Specifically, AccA might be a direct target of Fus3, which led to acetyl-CoA carboxylase activities were decreased in null-deletion and site mutagenesis strains. The results concluded that Fus3 could regulate the expression of acetyl-CoA and malonyl-CoA biosynthetic genes directly or indirectly, and then affect the AF production that relies on the regulation of AF substrate rather than the modulation of AF cluster genes. IMPORTANCE Aspergillus flavus is an important saprophytic fungus that produces aflatoxins (AF), which threaten food and feed safety. MAP (mitogen-activated protein) kanases are essential for fungal adaptation to diverse environments. Fus3, as the terminal kinase of a MAPK cascade, interacts with other MAPK modules and phosphorylates downstream targets. We provide evidence that Fus3 could affect AF biosynthesis by regulating the production of acetyl-CoA and malonyl-CoA, but this does not depend on the regulation of AF biosynthetic genes. Our results partly reveal the regulatory mechanism of Fus3 on AF biosynthesis and provide a novel AF modulation pattern, which may contribute to the discovery of new strategies in controlling A. flavus and AF contamination.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus flavus/enzimología , Proteínas Fúngicas/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Acetilcoenzima A/metabolismo , Secuencias de Aminoácidos , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Vías Biosintéticas , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/química , Proteínas Quinasas Activadas por Mitógenos/genética , Familia de Multigenes , Fosforilación , Unión ProteicaRESUMEN
BACKGROUND: L-Asparaginase is an antineoplastic agent used in the treatment of acute myeloid and acute lymphoblastic leukemia. The present study deals with the production of this chemotherapeutic enzyme drug from Aspergillus flavus NCIM 526. The production of enzymes was carried out using oil-extracted cakes in a shake flask culture. Process parameters like carbon and nitrogen sources were also taken into account. METHODS: A total of six isolates were used to screen out efficient microorganisms for enzyme production. Aspergillus flavus NCIM 526 exhibited 138 IU/ml of enzyme activity in oil extracted mix cake after 96 hours of the incubation period. Molasses and l-asparagine were proved to be the best carbon and nitrogen sources for enzyme production. The enzyme was purified by column chromatography and the finest enzyme exhibited specific activity of 28 IU/mg. RESULTS AND DISCUSSION: The fungal enzyme exhibited low Km values as compared with standard E. coli L-asparaginase, proving more substrate affinity of fungal enzyme than bacterial enzymes. CONCLUSION: The study explored the Aspergillus flavus NCIM 526 as a potential fungal source for high yield production of antileukemic enzyme drugs.
Asunto(s)
Asparaginasa , Aspergillus flavus/enzimología , Antineoplásicos/metabolismo , Asparaginasa/biosíntesis , Escherichia coliRESUMEN
The biological properties of elastase and Aspergillus flavus elastase inhibitor (AFLEI) from A. flavus were examined. Pathogenicity of elastase was investigated in mice immunocompromised with cyclophosphamide, cyclosporine, prednisolone and carrageenan. Compared to cyclophosphamide immunocompromised mice treated with the spores of elastase nonproducing strain, cyclophosphamide immunocompromised mice treated with the spores of elastase producing strain had a significantly shorter survival rate. Molecular mass of AFLEI was determined to be 7525.8 Da. The elastolytic activity of elastases from A. flavus, and human leukocytes were inhibited by AFLEI. The primary structure of AFLEI was determined by the Edman sequencing procedure. The search for amino acid homology with other proteins demonstrated that amino acid residues 1 to 68 of AFLEI are 100% identical to residues 20 to 87 of the hypothetical protein AFUA_3G14940 of A. fumigatus. When immunocompromised mice administered of cyclophosphamide were infected by inhalation of A. flavus then administered amphotericin B (AMPH) alone or in combination with AFLEI, survival rate tended to be higher with combination treatment than with AMPH alone. Moreover, although extensive bleeding was seen in pathology sections taken from rat lung resected 24 h after elastase was administered to the lung via the bronchus, this bleeding was inhibited by AFLEI. The X-ray analysis has revealed that the structure of this inhibitor was wedge shaped and composed of a binding loop and a scaffold protein core. As synthetic-inhibitor strongly inhibited cytotoxicity induced by elastase in human-derived cells, it could prove beneficial for the treatment of pulmonary aspergillosis.
Asunto(s)
Aspergillus flavus/química , Aspergillus flavus/patogenicidad , Inhibidores Enzimáticos/farmacología , Elastasa Pancreática/efectos adversos , Anfotericina B/administración & dosificación , Animales , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Hemorragia/tratamiento farmacológico , Hemorragia/etiología , Humanos , Huésped Inmunocomprometido , Enfermedades Pulmonares/tratamiento farmacológico , Enfermedades Pulmonares/etiología , Ratones , Elastasa Pancreática/química , Elastasa Pancreática/aislamiento & purificación , Aspergilosis Pulmonar/tratamiento farmacológico , RatasRESUMEN
Aspergillus flavus is one of the important human and plant pathogens causing not only invasive aspergillosis in immunocompromised patients but also crop contamination resulting from carcinogenic aflatoxins (AFs). Investigation of the targeting factors that are involved in pathogenicity is of unmet need to dismiss the hazard. Phosphoglucose isomerase (PGI) catalyzes the reversible conversion between glucose-6-phosphate and fructose-6-phosphate, thus acting as a key node for glycolysis, pentose phosphate pathway, and cell wall biosynthesis in fungi. In this study, we constructed an A. flavus pgi deletion mutant, which exhibited specific carbon requirement for survival, reduced conidiation, and slowed germination even under optimal experimental conditions. The Δpgi mutant lost the ability to form sclerotium and displayed hypersusceptibility to osmotic, oxidative, and temperature stresses. Furthermore, significant attenuated virulence of the Δpgi mutant was documented in the Caenorhabditis elegans infection model, Galleria mellonella larval model, and crop seeds. Our results indicate that PGI in A. flavus is a key enzyme in maintaining sugar homeostasis, stress response, and pathogenicity of A. flavus. Therefore, PGI is a potential target for controlling infection and AF contamination caused by A. flavus.
Asunto(s)
Aflatoxinas , Aspergillus flavus , Glucosa-6-Fosfato Isomerasa , Aspergillus flavus/enzimología , Aspergillus flavus/patogenicidad , Proteínas Fúngicas/genética , Glucosa-6-Fosfato Isomerasa/genética , Homeostasis , Azúcares , VirulenciaRESUMEN
S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) are widely distributed among almost all organisms and often characterized with conserved Rossmann fold, TIM barrel, and D×G×G×G motif. However, some MTases show no methyltransferase activity. In the present study, the crystal structure of LepI, one MTase-like enzyme isolated from A. flavus that catalyzes pericyclic reactions, was investigated to determine its structure-function relationship. The overall structure of LepI in complex with the SAM mimic S-adenosyl-L-homocysteine (SAH) (PDB ID: 6IV7) indicated that LepI is a tetramer in solution. The residues His133, Arg197, Arg295, and Asp296 located near the active site can form hydrogen bonds with the substrate, thus participating in catalytic reactions. The binding of SAH in LepI is almost identical to that in other resolved MTases; however, the location of catalytic residues differs significantly. Phylogenetic trials suggest that LepI proteins share a common ancestor in plants and algae, which may explain the conserved SAM-binding site. However, the accelerated evolution of A. flavus has introduced both functional and structural changes in LepI. More importantly, the residue Arg295, which is unique to LepI, might be a key determinant for the altered enzymatic behavior. Collectively, the differences in the composition of catalytic residues, as well as the unique tetrameric form of LepI, define its unique enzymatic behavior. The present work provides an additional understanding of the structure-function relationship of MTases and MTase-like enzymes.
Asunto(s)
Aspergillus flavus/enzimología , Proteínas Fúngicas/química , Metiltransferasas/química , S-Adenosilhomocisteína/química , S-Adenosilmetionina/química , Secuencia de Aminoácidos , Aspergillus flavus/química , Aspergillus flavus/clasificación , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Enlace de Hidrógeno , Metiltransferasas/genética , Metiltransferasas/metabolismo , Modelos Moleculares , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Aspergillus flavus (A. flavus) is one of the most important model environmental fungi which can produce a potent toxin and carcinogen known as aflatoxin. Aflatoxin contamination causes massive agricultural economic loss and a critical human health issue each year. Although a functional vacuole has been highlighted for its fundamental importance in fungal virulence, the molecular mechanisms of the vacuole in regulating the virulence of A. flavus remain largely unknown. Here, we identified a novel vacuole-related protein in A. flavus, the ortholog of phosphatidylinositol-3-phosphate-5-kinase (Fab1) in Saccharomyces cerevisiae. This kinase was located at the vacuolar membrane, and loss of fab1 function was found to affect the growth, conidia and sclerotial development, cellular acidification and metal ion homeostasis, aflatoxin production and pathogenicity of A. flavus. Further functional analysis revealed that Fab1 was required to maintain the vacuole size and cell morphology. Additional quantitative proteomic analysis suggested that Fab1 was likely to play an important role in maintaining vacuolar/cellular homeostasis, with vacuolar dysregulation upon fab1 deletion leading to impaired aflatoxin synthesis in this fungus. Together, these results provide insight into the molecular mechanisms by which this pathogen produces aflatoxin and mediates its pathogenicity, and may facilitate dissection of the vacuole-mediated regulatory network in A. flavus.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/genética , Aflatoxinas/biosíntesis , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Aflatoxinas/genética , Aspergillus flavus/patogenicidad , Proteínas Fúngicas/metabolismo , Homeostasis , Semillas/microbiología , Zea mays/microbiologíaRESUMEN
Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.
Asunto(s)
Aspergillus flavus , Endo-1,4-beta Xilanasas/biosíntesis , Proteínas Fúngicas/biosíntesis , Pectinas/metabolismo , Poligalacturonasa/biosíntesis , Xilanos/metabolismo , Aspergillus flavus/enzimología , Aspergillus flavus/crecimiento & desarrollo , Carbono/metabolismo , Especificidad de la EspecieRESUMEN
Aflatoxin contamination of crops is a serious problem worldwide. Utilization of aflatoxin production inhibitors is attractive, as the elucidation of their modes of action contributes to clarifying the mechanism of aflatoxin production. Here, we identified mitochondrial protease ClpP as the target of dioctatin, an inhibitor of aflatoxin production of Aspergillus flavus. Dioctatin conferred uncontrolled caseinolytic capacity on ClpP of A. flavus and Escherichia coli. Dioctatin-bound ClpP selectively degraded mitochondrial energy-related proteins in vitro, including a subunit of respiratory chain complex V, which was also reduced by dioctatin in a ClpP-dependent manner in vivo. Dioctatin enhanced glycolysis and alcohol fermentation while reducing tricarboxylic acid cycle metabolites. These disturbances were accompanied by reduced histone acetylation and reduced expression of aflatoxin biosynthetic genes. Our results suggest that dioctatin inhibits aflatoxin production by inducing ClpP-mediated degradation of mitochondrial energy-related components, and that mitochondrial energy metabolism functions as a key determinant of aflatoxin production.
Asunto(s)
Aflatoxinas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Ciclo del Ácido Cítrico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Mitocondrias/efectos de los fármacos , Aflatoxinas/biosíntesis , Aflatoxinas/genética , Aspergillus flavus/enzimología , Proteínas Bacterianas/metabolismo , Relación Dosis-Respuesta a Droga , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/química , Escherichia coli/enzimología , Mitocondrias/metabolismo , Estructura Molecular , Serina Endopeptidasas/metabolismoRESUMEN
Ascorbate oxidases are an enzyme group that has not been explored to a large extent. So far, mainly ascorbate oxidases from plants and only a few from fungi have been described. Although ascorbate oxidases belong to the well-studied enzyme family of multi-copper oxidases, their function is still unclear. In this study, Af_AO1, an enzyme from the fungus Aspergillus flavus, was characterized. Sequence analyses and copper content determination demonstrated Af_AO1 to belong to the multi-copper oxidase family. Biochemical characterization and 3D-modeling revealed a similarity to ascorbate oxidases, but also to laccases. Af_AO1 had a 10-fold higher affinity to ascorbic acid (KM = 0.16 ± 0.03 mM) than to ABTS (KM = 1.89 ± 0.12 mM). Furthermore, the best fitting 3D-model was based on the ascorbate oxidase from Cucurbita pepo var. melopepo. The laccase-like activity of Af_AO1 on ABTS (Vmax = 11.56 ± 0.15 µM/min/mg) was, however, not negligible. On the other hand, other typical laccase substrates, such as syringaldezine and guaiacol, were not oxidized by Af_AO1. According to the biochemical and structural characterization, Af_AO1 was classified as ascorbate oxidase with unusual, laccase-like activity.
Asunto(s)
Ascorbato Oxidasa/metabolismo , Aspergillus flavus/enzimología , Lacasa/metabolismo , Secuencia de Aminoácidos , Ascorbato Oxidasa/química , Cobre/metabolismo , Cinética , Lacasa/química , Modelos Moleculares , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Reactive oxygen species (ROS) induce the synthesis of a myriad of secondary metabolites, including aflatoxins. It raises significant concern as it is a potent environmental contaminant. In Aspergillus flavus., antioxidant enzymes link ROS stress response with coordinated gene regulation of aflatoxin biosynthesis. In this study, we characterized the function of a core component of the antioxidant enzyme catalase (CTA1) of A. flavus. Firstly, we verified the presence of cta1 corresponding protein (CTA1) by Western blot analysis and mass-spectrometry based analysis. Then, the functional study revealed that the growth, sporulation and sclerotia formation significantly increased, while aflatoxins production and virulence were decreased in the cta1 deletion mutant as compared with the WT and complementary strains. Furthermore, the absence of the cta1 gene resulted in a significant rise in the intracellular ROS level, which in turn added to the oxidative stress level of cells. A further quantitative proteomics investigation hinted that in vivo, CTA1 might maintain the ROS level to facilitate the aflatoxin synthesis. All in all, the pleiotropic phenotype of A. flavus CTA1 deletion mutant revealed that the antioxidant system plays a crucial role in fungal development, aflatoxins biosynthesis and virulence.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus flavus/enzimología , Aspergillus flavus/patogenicidad , Catalasa/metabolismo , Virulencia/genética , Antioxidantes/metabolismo , Aspergillus flavus/genética , Catalasa/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismo , Eliminación de Secuencia , Virulencia/efectos de los fármacosRESUMEN
Gout is a common rheumatic condition caused due to increase in serum uric acid level (hyperuricemia). Uricase is for lowering the level of uric acid but unfortunately, it is not produced in humans due to evolutionary changes. Therefore, it is administered to humans from outside in case of the high uric acid level in blood. A different formulation of uricase from bacterial, fungal, and mammalian sources is present in the market for the treatment of hyperuricemia conditions. Uricase formulation showed immunogenic response due to the occurrence of hypersensitivity reaction during the treatment that results in poor patient compliance. The purpose of this study was to clarify the variation of Uricase immunogenicity from different sources. We have used some immunoinformatic approaches to analyze and understand some structural aspects of immunogenic and allergenic epitopes of Uricase by calculation of relative frequency for eleven global alleles. As per our knowledge, this is the first immunoinformatic study of Uricase (structural based immunogenicity prediction) that deciphered the high immunogenic nature of Uricase but no significant difference in immunogenicity was found among Uricase isolated from Aspergillus flavus, Bacillus subtillis, and mammalian source. This study gives a further lead to develop some methods (include bioengineering of less immunogenic version of the uricase or utilizing the homologous enzymes) for minimizing immune response or search new sources of uricase that could be less or non-immunogenic.
Asunto(s)
Epítopos de Linfocito B/química , Epítopos de Linfocito T/química , Urato Oxidasa , Animales , Aspergillus flavus/enzimología , Bacillus subtilis/enzimología , Biología Computacional , Mapeo Epitopo , Gota/tratamiento farmacológico , Gota/inmunología , Humanos , Hiperuricemia/tratamiento farmacológico , Hiperuricemia/inmunología , Mamíferos , Conformación Proteica , Urato Oxidasa/química , Urato Oxidasa/inmunologíaRESUMEN
Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.
Asunto(s)
Aspergillus flavus/genética , Aspergillus oryzae/genética , Genoma Fúngico/genética , Genómica , Aspergillus flavus/clasificación , Aspergillus flavus/enzimología , Aspergillus oryzae/clasificación , Aspergillus oryzae/enzimología , Reactores Biológicos , Metabolismo de los Hidratos de Carbono/genética , Productos Agrícolas/microbiología , ADN de Hongos/genética , Fermentación , Alimentos Fermentados , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Redes y Vías Metabólicas/genética , Familia de Multigenes , Fenotipo , Filogenia , Enfermedades de las Plantas/prevención & control , Metabolismo Secundario/genéticaRESUMEN
Aspergillus flavus represents an important fungal pathogen, causing severe economic losses in crops. The mitogen-activated protein (MAP) kinase signaling pathway contributes to many physiological processes, but its precise role in A. flavus is not yet fully understood. In this study, we focused on the AflBck1 gene, which encodes a MAP kinase kinase kinase of the Slt2-MAPK pathway. Targeted deletion of AflBck1 led to a significant defect in growth and development, and a AflBck1-deleted mutant (∆AflBck1) showed higher sensitivity to cell-wall stress than wild type (WT). Importantly, we observed that ∆AflBck1 displayed an enhanced ability to produce aflatoxin, a potential carcinogenic mycotoxin. However, the pathogenicity of the ∆AflBck1 mutant was markedly reduced in peanut seeds. We also presented evidence that AflBck1 was genetically epistatic to AflMkk2 in the Slt2-MAPK pathway. Finally, we found that loss of the proline-rich region at the N terminus of AflBck1 affected the reproduction of A. flavus. Collectively, this study not only extended the understanding that the MAPK pathway regulated A. flavus pathogenicity but also provided a possible strategy to control A. flavus contamination.
Asunto(s)
Aspergillus flavus , Pared Celular , Proteínas Fúngicas , Virulencia , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Aspergillus flavus/crecimiento & desarrollo , Aspergillus flavus/patogenicidad , Pared Celular/enzimología , Quinasas Quinasa Quinasa PAM/genética , Virulencia/genéticaRESUMEN
The aim of this work was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as estimated by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards L-glutamine as substrate, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml-1. The enzyme was activated by Na+ and Co2+, while it was greatly suppressed by iodoacetate, NEM, Zn2+ and Hg2+ at 10 mM. L-glutaminase activity increased with a gradual increase of sodium chloride concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither a cognizable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic impact on Hela and Hep G2 cell lines with an IC50 value 18 and 12 µg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 µg/ml, respectively.
Asunto(s)
Antineoplásicos/farmacología , Aspergillus flavus/enzimología , Glutaminasa/farmacología , Animales , Antineoplásicos/aislamiento & purificación , Plaquetas/efectos de los fármacos , Estabilidad de Enzimas , Glutaminasa/aislamiento & purificación , Células HeLa , Células Hep G2 , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Cinética , Dosificación Letal Mediana , Peso Molecular , Ratas , Ratas Sprague-Dawley , Especificidad por SustratoRESUMEN
Aspergillus flavus is a pathogenic fungus that produces carcinogenic aflatoxins, posing a great threat to crops, animals and humans. Lysine acetylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. However, current information on the extent and function of lysine acetylation and aflatoxin biosynthesis in A. flavus is limited. Here, a global acetylome analysis of A. flavus was performed by peptide pre-fractionation, pan-acetylation antibody enrichment and liquid chromatography-mass spectrometry. A total of 1313 high-confidence acetylation sites in 727 acetylated proteins were identified in A. flavus. These acetylation proteins are widely involved in glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle and aflatoxin biosynthesis. AflO (O-methyltransferase), a key enzyme in aflatoxin biosynthesis, was found to be acetylated at K241 and K384. Deletion of aflO not only impaired conidial and sclerotial developments, but also dramatically suppressed aflatoxin production and pathogenicity of A. flavus. Further site-specific mutations showed that lysine acetylation of AflO could also result in defects in development, aflatoxin production and pathogenicity, suggesting that acetylation plays a vital role in the regulation of the enzymatic activity of AflO in A. flavus. Our findings provide evidence for the involvement of lysine acetylation in various biological processes in A. flavus and facilitating in the elucidation of metabolic networks.
Asunto(s)
Aflatoxinas/biosíntesis , Aspergillus flavus/enzimología , Aspergillus flavus/patogenicidad , Proteínas Fúngicas/metabolismo , Lisina/metabolismo , Metiltransferasas/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Acetilación , Arachis/microbiología , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Ciclo del Ácido Cítrico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Espectrometría de Masas , Redes y Vías Metabólicas , Metiltransferasas/química , Metiltransferasas/genética , Vía de Pentosa Fosfato , Enfermedades de las Plantas/microbiología , Procesamiento Proteico-Postraduccional , Esporas Fúngicas/genética , Esporas Fúngicas/metabolismo , Esporas Fúngicas/patogenicidad , VirulenciaRESUMEN
Alginate- polyethyleneimine gel beads modified by using 0.3â¯M Na+ were used for covalent immobilization of Aspergillus flavus xylanase. SEM images showed distorted structure with addition of Na+ that impaired the egg-box structure formation offered much covalent binding with xylanase. Immobilization onto (Alg+PEI/Na+) showed an enhancement in the operational stability, immobilization efficiency as well as immobilization yield. Covalent immobilization of xylanase onto (Alg+PEI/Na+) enhanced xylanase activity over a wide range of pHs (4-5.5) comparable to its free formula. As well as an increase in reaction temperature up to 60°C. However, immobilized formula of enzyme showed abroad thermal stability that it retained 79.0% of its initial activity at 70°C up to 30â¯min whereas, free formula completely lost its activity at this temperature. Thermodynamics studies showed an enhancement in thermal stability at high temperature for the immobilized xylanase. i.e. At 70°C the t1/2 and D-value for free formula of enzyme increased from 24 to165 min and from 79.95to 548.23â¯min, respectively. Moreover, the enzyme stability enhancement for immobilized formula of xylanase was proved with a remarkable increase in enthalpy and free energy. 93% of the immobilized xylanase activity was retained over 6â¯weeks of storage at -4°C.
Asunto(s)
Alginatos/química , Endo-1,4-beta Xilanasas/metabolismo , Enzimas Inmovilizadas/metabolismo , Polietileneimina/química , Sodio/química , Aspergillus flavus/enzimología , Biocatálisis , Cationes , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Microesferas , Espectrometría por Rayos X , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , Xilanos/metabolismoRESUMEN
Amongst the various approaches to contain aflatoxin contamination of feed and food commodities, the use of inhibitors of fungal growth and/or toxin biosynthesis is showing great promise for the implementation or the replacement of conventional pesticide-based strategies. Several inhibition mechanisms were found taking place at different levels in the biology of the aflatoxin-producing fungal species such as Aspergillus flavus: compounds that influence aflatoxin production may block the biosynthetic pathway through the direct control of genes belonging to the aflatoxin gene cluster, or interfere with one or more of the several steps involved in the aflatoxin metabolism upstream. Recent findings pointed to mitochondrial functionality as one of the potential targets of some aflatoxin inhibitors. Additionally, we have recently reported that the effect of a compound belonging to the class of thiosemicarbazones might be related to the energy generation/carbon flow and redox homeostasis control by the fungal cell. Here, we report our investigation about a putative molecular target of the 3-isopropylbenzaldehyde thiosemicarbazone (mHtcum), using the yeast Saccharomyces cerevisiae as model system, to demonstrate how the compound can actually interfere with the mitochondrial respiratory chain.
Asunto(s)
Aflatoxinas/antagonistas & inhibidores , Antifúngicos/farmacología , Regulación Fúngica de la Expresión Génica , Mitocondrias/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Tiosemicarbazonas/farmacología , Aflatoxinas/biosíntesis , Antifúngicos/química , Aspergillus flavus/efectos de los fármacos , Aspergillus flavus/enzimología , Aspergillus flavus/genética , Sitios de Unión , Transporte de Electrón/efectos de los fármacos , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Modelos Biológicos , Simulación del Acoplamiento Molecular , Familia de Multigenes , Unión Proteica , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Tiosemicarbazonas/químicaRESUMEN
Therapeutic proteins are indispensable in the treatment of various human diseases. Despite the many benefits of therapeutic proteins, they also exhibit diverse side effects. Therefore, reducing unwanted side effects of therapeutic proteins as well as enhancing their therapeutic efficacy are very important in developing therapeutic proteins. Urate oxidase (UOX) is a therapeutic enzyme that catalyzes the conversion of uric acid (UA) into a soluble metabolite, and it is used clinically for the treatment of hyperuricemia. Since UA degradation by UOX generates H2O2 (a cytotoxic side product), UOX was co-delivered with catalase-mimic nanoparticles (AuNPs) using biocompatible pluronic-based nanocarriers (NCs) to effectively reduce H2O2-associated toxicity in cultured cells and to enhance UA degradation efficiency in vivo. Simple temperature-dependent size changes of NCs allowed co-encapsulation of both UOX and AuNPs at a high loading efficiency without compromising critical properties, resulting in efficient modulation of a mixing ratio of UOX and AuNPs encapsulated in NCs. Co-localizing UOX and AuNPs in the NCs led to enhanced UA degradation and H2O2 removal in vitro, leading to a great reduction in H2O2-associated cytotoxicity compared with UOX alone or a free mixture of UOX and AuNPs. Furthermore, we demonstrated that co-delivery of UOX and AuNPs using NCs significantly improves in vivo UA degradation compared to simple co-injection of free UOX and AuNPs. More broadly, we showed that biocompatible pluronic-based nanocarriers can be used to deliver a target therapeutic protein along with its toxicity-eliminating agent in order to reduce side effects and enhance efficacy.