RESUMO
Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.
Assuntos
Aflatoxinas , Aspergillus flavus , Aflatoxinas/metabolismo , Aspergillus/metabolismo , Aspergillus flavus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Metabolismo Secundário , Esporos FúngicosRESUMO
Post-translational modifications (PTMs) are important for protein function and regulate multiple cellular processes and secondary metabolites (SMs) in fungi. Aspergillus species belong to a genus renown for an abundance of bioactive secondary metabolites, many important as toxins, pharmaceuticals and in industrial production. The genes required for secondary metabolites are typically co-localized in biosynthetic gene clusters (BGCs), which often localize in heterochromatic regions of genome and are 'turned off' under laboratory condition. Efforts have been made to 'turn on' these BGCs by genetic manipulation of histone modifications, which could convert the heterochromatic structure to euchromatin. Additionally, non-histone PTMs also play critical roles in the regulation of secondary metabolism. In this review, we collate the known roles of epigenetic and PTMs on Aspergillus SM production. We also summarize the proteomics approaches and bioinformatics tools for PTM identification and prediction and provide future perspectives on the emerging roles of PTM on regulation of SM biosynthesis in Aspergillus and other fungi.
Assuntos
Aspergillus , Família Multigênica , Aspergillus/genética , Aspergillus/metabolismo , Fungos/genética , Processamento de Proteína Pós-Traducional , Metabolismo Secundário/genéticaRESUMO
Aspergillus flavus is an opportunistic fungal pathogen that colonizes agriculture crops with aflatoxin contamination. We found that Perillaldehyde (PAE) effectively inhibited A. flavus viability and aflatoxin production by inducing excess reactive oxygen species (ROS). Transcriptome analysis indicated that the Gα protein FadA was significantly induced by PAE. Functional characterization of FadA showed it is important for asexual development and aflatoxin biosynthesis by regulation of cAMP-PKA signalling. The ΔfadA mutant was more sensitive to PAE, while ΔpdeL and ΔpdeH mutants can tolerate excess PAE compared to wild-type A. flavus. Further RNA-sequence analysis showed that fadA was important for expression of genes involved in oxidation-reduction and cellular metabolism. The flow cytometry and fluorescence microscopy demonstrated that ΔfadA accumulated more concentration of ROS in cells, and the transcriptome data indicated that genes involved in ROS scavenging were downregulated in ΔfadA mutant. We further found that FadA participated in regulating response to extracellular environmental stresses by increasing phosphorylation levels of MAPK Kinase Slt2 and Hog1. Overall, our results indicated that FadA signalling engages in mycotoxin production and A. flavus resistance to antimicrobial PAE, which provide valuable information for controlling this fungus and AF biosynthesis in pre- and postharvest of agricultural crops.
Assuntos
Aflatoxinas , Anti-Infecciosos , Anti-Infecciosos/metabolismo , Aspergillus flavus/metabolismo , Produtos Agrícolas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Monoterpenos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Heterotrimeric G-proteins play crucial roles in growth, asexual development, and pathogenicity of fungi. The regulator of G-protein signaling (RGS) proteins function as negative regulators of the G proteins to control the activities of GTPase in Gα subunits. In this study, we functionally characterized the six RGS proteins (i.e., RgsA, RgsB, RgsC, RgsD, RgsE, and FlbA) in the pathogenic fungus Aspergillus flavus. All the aforementioned RGS proteins were also found to be functionally different in conidiation, aflatoxin (AF) biosynthesis, and pathogenicity in A. flavus. Apart from FlbA, all other RGS proteins play a negative role in regulating both the synthesis of cyclic AMP (cAMP) and the activation of protein kinase A (PKA). Additionally, we also found that although RgsA and RgsE play a negative role in regulating the FadA-cAMP/PKA pathway, they function distinctly in aflatoxin biosynthesis. Similarly, RgsC is important for aflatoxin biosynthesis by negatively regulating the GanA-cAMP/PKA pathway. PkaA, which is the cAMP-dependent protein kinase catalytic subunit, also showed crucial influences on A. flavus phenotypes. Overall, our results demonstrated that RGS proteins play multiple roles in the development, pathogenicity, and AF biosynthesis in A. flavus through the regulation of Gα subunits and cAMP-PKA signals. IMPORTANCE RGS proteins, as crucial regulators of the G protein signaling pathway, are widely distributed in fungi, while little is known about their roles in Aspergillus flavus development and aflatoxin. In this study, we identified six RGS proteins in A. flavus and revealed that these proteins have important functions in the regulation of conidia, sclerotia, and aflatoxin formation. Our findings provide evidence that the RGS proteins function upstream of cAMP-PKA signaling by interacting with the Gα subunits (GanA and FadA). This study provides valuable information for controlling the contamination of A. flavus and mycotoxins produced by this fungus in pre- and postharvest of agricultural crops.
Assuntos
Aflatoxinas , Proteínas RGS , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas RGS/genética , Proteínas RGS/metabolismo , Transdução de Sinais/genética , Esporos FúngicosRESUMO
Monoterpene Perillaldehyde (PAE) is a major component of the essential oil extracted from perilla plants (Perilla frutescens), which has been used as a leafy vegetable and a medicinal agent. PAE has gained a lot of attention in recent years because of its antifungal and other microbial activities and, human health benefits. PAE has also been used as food additives, perfume ingredients, and traditional medicine concoctions. Biological analyses of PAE have revealed that it has good antioxidant activities and can serve as organic fruit and food preservative. Animal studies indicated potent anticancer, anti-depressant, and anti-inflammatory effects of PAE. Also, PAE is certified "generally recognized as safe" (GRAS) and not mutagenic. However, moderation during usage is advisable, as minor adverse effects are associated with a very high dosage. Despite the newly reported findings, its properties have not been thoroughly summarized and reviewed. Also, clinical trials and official large-scale field applications of PAE in the agricultural sectors are yet to be reported. In this review, updated PAE research progress was provided, focusing on its antifungal and other antimicrobial properties and the mechanisms behind it, phytochemical profile, pharmacological effects, and safety concerns.HighlightsIsolation and recovery techniques of PAE from perilla plants have been developed and improved in recent years.PAE is a potential anti-oxidant and antifungal agent that can be widely used in the food industry.PAE can be developed into drug ingredients for pharmaceutical industries due to its anti-inflammatory, anti-cancer and anti-depressant activities.PAE can be safely used in human when low and moderate dosage is used.
Assuntos
Perilla , Animais , Anti-Inflamatórios/farmacologia , Antifúngicos/farmacologia , Humanos , Monoterpenos , Perilla/químicaRESUMO
The fundamental biological function of nucleoside diphosphate kinase (NDK) is to catalyze the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP). This kinase also has functions that extend beyond its canonically defined enzymatic role as a phosphotransferase. However, the role of NDK in filamentous fungi, especially in Aspergillus flavus (A. flavus), is not yet known. Here we report that A. flavus has two NDK-encoding gene copies as assessed by qPCR. Using gene-knockout and complementation experiments, we found that AfNDK regulates spore and sclerotia development and is involved in plant virulence as assessed in corn and peanut seed-based assays. An antifungal test with the inhibitor azidothymidine suppressed AfNDK activity in vitro and prevented spore production and sclerotia formation in A. flavus, confirming AfNDK's regulatory functions. Crystallographic analysis of AfNDK, coupled with site-directed mutagenesis experiments, revealed three residues (Arg-104, His-117, and Asp-120) as key sites that contribute to spore and sclerotia development. These results not only enrich our knowledge of the regulatory role of this important protein in A. flavus, but also provide insights into the prevention of A. flavus infection in plants and seeds, as well as into the structural features relevant for future antifungal drug development.
Assuntos
Aspergillus flavus/enzimologia , Proteínas Fúngicas , Núcleosídeo-Difosfato Quinase , Esporos Fúngicos/enzimologia , Fatores de Virulência , Arachis/microbiologia , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Núcleosídeo-Difosfato Quinase/química , Núcleosídeo-Difosfato Quinase/metabolismo , Sementes/microbiologia , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Zea mays/microbiologiaRESUMO
Candida albicans invasion is one of the most serious fungal infections in clinical history. In recent years, because of the widespread use of immunosuppressive drugs, chemotherapy drugs, glucocorticoids, and broad-spectrum antibiotics, serious drug resistance has been reported; therefore, a new type of antifungal drug needs to be developed. In this study, we found that Nerol (NEL) had strong antimicrobial activity and 0.77 µL/mL NEL was the minimum inhibitory concentration (MIC) effective against C. albicans. We determined the change of the growth curve of NEL for C. albicans, to identify the trend of NEL activity against C. albicans. Through the determination of the ergosterol content and glucose-induced extracellular fluid acidification of NEL on C. albicans, we found that NEL inhibits the growth of C. albicans by destroying cell membranes. This finding was also supported by the expression of SAP (secreted aspartyl proteinase) involved in cell membrane synthesis. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and PAS (periodic acid-Schiff) staining were conducted to prove that NEL had the ability to treated mouse oral C. albicans infection and vaginal C. albicans infection. This research may help us to investigate new antimicrobial agents for treating C. albicans infections. KEY POINTS: ⢠NEL can inhibit the growth of C. albicans. ⢠NEL destroys the cell membrane formation and permeability of C. albicans. ⢠NEL can treat vulvovaginal candidiasis and oropharyngeal candidiasis in mice. ⢠NEL could be used as a possible antifungal agent.
Assuntos
Monoterpenos Acíclicos/uso terapêutico , Antifúngicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candidíase Vulvovaginal/tratamento farmacológico , Doenças da Boca/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Animais , Ácido Aspártico Proteases/genética , Candida albicans/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , Candidíase Vulvovaginal/microbiologia , Membrana Celular/efeitos dos fármacos , Ergosterol/análise , Feminino , Masculino , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Boca/microbiologia , Doenças da Boca/microbiologiaRESUMO
Aflatoxins (AFs) have always been regarded as the most effective carcinogens, posing a great threat to agriculture, food safety, and human health. Aspergillus flavus is the major producer of aflatoxin contamination in crops. The prevention and control of A. flavus and aflatoxin continues to be a global problem. In this study, we demonstrated that the cell-free culture filtrate of Aspergillus oryzae and a non-aflatoxigenic A. flavus can effectively inhibit the production of AFB1 and the growth and reproduction of A. flavus, indicating that both of the non-aflatoxigenic Aspergillus strains secrete inhibitory compounds. Further transcriptome sequencing was performed to analyze the inhibitory mechanism of A. flavus treated with fermenting cultures, and the results revealed that genes involved in the AF biosynthesis pathway and other biosynthetic gene clusters were significantly downregulated, which might be caused by the reduced expression of specific regulators, such as AflS, FarB, and MtfA. The WGCNA results further revealed that genes involved in the TCA cycle and glycolysis were potentially involved in aflatoxin biosynthesis. Our comparative transcriptomics also revealed that two conidia transcriptional factors, brlA and abaA, were found to be significantly downregulated, which might lead to the downregulation of conidiation-specific genes, such as the conidial hydrophobins genes rodA and rodB. In summary, our research provides new insights for the molecular mechanism of controlling AF synthesis to control the proliferation of A. flavus and AF pollution.
Assuntos
Aflatoxinas , Aspergillus flavus/fisiologia , Regulação Fúngica da Expressão Gênica , RNA-Seq , Esporos Fúngicos , Transcriptoma , Aflatoxinas/biossíntese , Aflatoxinas/genética , Glycine max/microbiologia , Esporos Fúngicos/genética , Esporos Fúngicos/metabolismoRESUMO
Perillaldehyde (PAE), an essential oil in Perilla plants, serves as a safe flavor ingredient in foods, and shows an effectively antifungal activity. Reactive oxygen species (ROS) accumulation in Aspergillus flavus plays a critical role in initiating a metacaspase-dependent apoptosis. However, the reason for ROS accumulation in A. flavus is not yet clear. Using transcriptome sequencing of A. flavus treated with different concentrations of PAE, our data showed that the ROS accumulation might have been as a result of an inhibition of energy metabolism with less production of reducing power. By means of GO and KEGG enrichment analysis, we screened four key pathways, which were divided into two distinct groups: a downregulated group that was made up of the glycolysis and pentose phosphate pathway, and an upregulated group that consisted of MAPK signaling pathway and GSH metabolism pathway. The inhibition of dehydrogenase gene expression in two glycometabolism pathways might play a crucial role in antifungal mechanism of PAE. Also, in our present study, we systematically showed a gene interaction network of how genes of four subsets are effected by PAE stress on glycometabolism, oxidant damage repair, and cell cycle control. This research may contribute to explaining an intrinsic antifungal mechanism of PAE against A. flavus.
Assuntos
Antifúngicos/farmacologia , Aspergillus flavus/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Monoterpenos/farmacologia , Transcriptoma/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Aspergillus flavus/genética , Aspergillus flavus/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Monoterpenos/metabolismo , Óleos Voláteis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Mapas de Interação de Proteínas , Espécies Reativas de Oxigênio/metabolismoRESUMO
Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.
Assuntos
Aflatoxinas , Aspergillus flavus , Regulação Fúngica da Expressão Gênica , Histona Desacetilases , Aflatoxinas/biossíntese , Aflatoxinas/genética , Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Aspergillus flavus/patogenicidade , Regulação Fúngica da Expressão Gênica/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Ligação Proteica , Virulência/genéticaRESUMO
Aspergillus flavus, a ubiquitous filamentous fungus found in soil, plants and other substrates has been reported not only as a pathogen for plants, but also a carcinogen producing fungus for human. Peptidyl-Prolyl Isomerase (PPIases) plays an important role in cell process such as protein secretion cell cycle control and RNA processing. However, the function of PPIase has not yet been identified in A. flavus. In this study, the PPIases gene from A. flavus named ppci1 was cloned into expression vector and the protein was expressed in prokaryotic expression system. Activity of recombinant ppci1 protein was particularly inhibited by FK506, CsA and rapamycin. 3D-Homology model of ppci1 has been constructed with the template, based on 59.7% amino acid similarity. The homologous recombination method was used to construct the single ppci1 gene deletion strain Δppci1. We found that, the ppci1 gene plays important roles in A. flavus growth, conidiation, and sclerotia formation, all of which showed reduction in Δppci1 and increased in conidiation compared with the wild-type and complementary strains in A. flavus. Furthermore, aflatoxin and peanut seeds infection assays indicated that ppci1 contributes to virulence of A. flavus. Furthermore, we evaluated the effect of PPIase inhibitors on A. flavus growth, whereby these were used to treat wild-type strains. We found that the growths were inhibited under every inhibitor. All, these results may provide valuable information for designing inhibitors in the controlling infections of A. flavus.
Assuntos
Aspergillus flavus/enzimologia , Aspergillus flavus/genética , Peptidilprolil Isomerase/genética , Sequência de Aminoácidos , Biologia Computacional/métodos , Espectrometria de Massas , Simulação de Dinâmica Molecular , Peptídeos , Peptidilprolil Isomerase/química , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/metabolismo , Filogenia , Conformação Proteica , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Cyclic AMP signaling controls a range of physiological processes in response to extracellular stimuli in organisms. Among the signaling cascades, cAMP, as a second messenger, is orchestrated by adenylate cyclase (biosynthesis) and cAMP phosphodiesterases (PDEs) (hydrolysis). In this study, we investigated the function of the high-affinity (PdeH) and low-affinity (PdeL) cAMP phosphodiesterase from the carcinogenic aflatoxin producing fungus Aspergillus flavus, and found that instead of PdeL, inactivation of PdeH exhibited a reduction in conidiation and sclerotia formation. However, the ΔpdeL/ΔpdeH mutant exhibited an enhanced phenotype defects, a similar phenotype defects to wild-type strain treated with exogenous cAMP. The activation of PKA activity was inhibited in the ΔpdeH or ΔpdeL/ΔpdeH mutant, both of whom exhibited increasing AF production. Further analysis by qRT-PCR revealed that pdeH had a high transcriptional level compared to pdeL in wild-type strain, and affected pdeL transcription. Green fluorescent protein tagging at the C-terminus of PDEs showed that PdeH-GFP is broadly compartmentalized in the cytosol, while PdeL-GFP localized mainly to the nucleus. Overall, our results indicated that PdeH plays a major role, but has overlapping function with PdeL, in vegetative growth, development and AF biosynthesis in A. flavus.
Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/genética , Diester Fosfórico Hidrolases/genética , Esporos Fúngicos/genética , Aflatoxinas/genética , Núcleo Celular/genética , AMP Cíclico , Regulação Fúngica da Expressão Gênica , Transdução de Sinais , Esporos Fúngicos/crescimento & desenvolvimentoRESUMO
Small non-coding RNA (sRNA) in various organisms remains a mysterious subject. Although microRNAs (miRNAs) have been intensively investigated in plants and animals, the study of miRNAs in fungi has been limited. Only microRNA-like RNAs (milRNAs) have been reported in several filamentous fungi. In this study, Illumina deep sequencing was performed to characterize the sRNA in Aspergillus flavus and to evaluate their responses to water activity and temperature. Global expression analysis showed an extensively differential expression of sRNA loci in A. flavus under different temperature or water activities. In addition, a total of 135 milRNAs were identified in A. flavus. The milRNA profiles obtained in deep sequencing were further validated by RT-qPCR assay. The presence and differential expression of milRNAs under different temperature or water activities in A. flavus imply that milRNAs might play important roles in the mycotoxin biosynthesis and mycelium growth in fungi A. flavus.
Assuntos
Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/efeitos da radiação , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos da radiação , MicroRNAs/biossíntese , Temperatura , Água/metabolismo , Aspergillus flavus/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Contamination of crop seeds and feed with Aspergillus flavus and its associated aflatoxins presents a significant threat to human and animal health due to their hepatotoxic and carcinogenic properties. To address this challenge, researchers have screened for potential biological control agents in peanut soil and pods. This study identified a promising candidate, a strain of the nonpigmented bacterium, Achromobacter xylosoxidans ZJS2-1, isolated from the peanut rhizosphere in Zhejiang Province, China, exhibiting notable antifungal and antiaflatoxin activities. Further investigations demonstrated that ZJS2-1 active substances (ZAS) effectively inhibited growth at a MIC of 60 µL/mL and nearly suppressed AFB1 production by 99%. Metabolomic analysis revealed that ZAS significantly affected metabolites involved in cell wall and membrane biosynthesis, leading to compromised cellular integrity and induced apoptosis in A. flavus through the release of cytochrome c. Notably, ZAS targeted SrbA, a key transcription factor involved in ergosterol biosynthesis and cell membrane integrity, highlighting its crucial role in ZJS2-1's biocontrol mechanism. Moreover, infection of crop seeds and plant wilt caused by A. flavus can be efficiently alleviated by ZAS. Additionally, ZJS2-1 and ZAS demonstrated significant inhibitory effects on various Aspergillus species, with inhibition rates ranging from 80 to 99%. These findings highlight the potential of ZJS2-1 as a biocontrol agent against Aspergillus species, offering a promising solution to enhance food safety and protect human health.
Assuntos
Achromobacter denitrificans , Aflatoxinas , Apoptose , Arachis , Aspergillus flavus , Membrana Celular , Rizosfera , Aspergillus flavus/metabolismo , Aspergillus flavus/crescimento & desenvolvimento , Arachis/microbiologia , Arachis/química , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Aflatoxinas/biossíntese , Aflatoxinas/metabolismo , Apoptose/efeitos dos fármacos , Achromobacter denitrificans/metabolismo , Sementes/microbiologia , Sementes/química , Sementes/metabolismo , Antifúngicos/farmacologia , Antifúngicos/metabolismo , China , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Microbiologia do SoloRESUMO
Mechanical forces play a crucial role in cellular processes, including ferroptosis, a form of regulated cell death associated with various diseases. However, the mechanical aspects of organelle lipid droplets (LDs) during ferroptosis are poorly understood. In this study, we designed and synthesized a fluorescent probe, TPE-V1, to enable real-time monitoring of LDs' viscosity using a dual-channel fluorescence-on model (red channel at 617 nm and NIR channel at 710 nm). The fluorescent imaging of using TPE-V1 was achieved due to the integrated mechanisms of the twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE). Through dual-emission channel fluorescence imaging, we observed the enhanced mechanical energy of LDs triggering cellular mechanosensing, including ferroptosis and cell deformation. Theoretical calculations confirmed the probe's behavior, showing that high-viscosity media prevented the rotation processes and restored fluorescence quenching in low viscosity. These findings suggest that our TICT-TPE design strategy provides a practical approach to study LDs' mechanical properties during ferroptosis. This development enhances our understanding of the interplay between mechanical forces and LDs, contributing to the knowledge of ferroptotic cell death and potential therapeutic interventions targeting dysregulated cell death processes.
Assuntos
Ferroptose , Corantes Fluorescentes , Gotículas Lipídicas , Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Corantes Fluorescentes/química , Humanos , Imagem Óptica , Viscosidade , FluorescênciaRESUMO
Nitric oxide (NO) is a signaling molecule with diverse roles in various organisms. However, its role in the opportunistic pathogen Aspergillus flavus remains unclear. This study investigates the potential of NO, mediated by metabolites from A. oryzae (AO), as an antifungal strategy against A. flavus. We demonstrated that AO metabolites effectively suppressed A. flavus asexual development, a critical stage in its lifecycle. Transcriptomic analysis revealed that AO metabolites induced NO synthesis genes, leading to increased intracellular NO levels. Reducing intracellular NO content rescued A. flavus spores from germination inhibition caused by AO metabolites. Furthermore, exogenous NO treatment and dysfunction of flavohemoglobin Fhb1, a key NO detoxification enzyme, significantly impaired A. flavus asexual development. RNA-sequencing and metabolomic analyses revealed significant metabolic disruptions within tricarboxylic acid (TCA) cycle upon AO treatment. NO treatment significantly reduced mitochondrial membrane potential (Δψm) and ATP generation. Additionally, aberrant metabolic flux within the TCA cycle was observed upon NO treatment. Further analysis revealed that NO induced S-nitrosylation of five key TCA cycle enzymes. Genetic analysis demonstrated that the S-nitrosylated Aconitase Acon and one subunit of succinate dehydrogenase Sdh2 played crucial roles in A. flavus development by regulating ATP production. This study highlights the potential of NO as a novel antifungal strategy to control A. flavus by compromising its mitochondrial function and energy metabolism.
Assuntos
Aspergillus flavus , Ciclo do Ácido Cítrico , Mitocôndrias , Óxido Nítrico , Ciclo do Ácido Cítrico/efeitos dos fármacos , Aspergillus flavus/metabolismo , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/efeitos dos fármacos , Óxido Nítrico/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Antifúngicos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.
RESUMO
Root rot caused by Fusarium solani is one of major postharvest diseases limiting sweet potato production. Antifungal effect and possible mode of action of cinnamaldehyde (CA) against F. solani were investigated. CA concentration of 0.075 g/L inhibited conidial viability of F. solani. CA vapor of 0.3 g/L in air completely controlled the F. solani development in sweet potatoes during storage for 10 days at 28 °C, and protected soluble sugar and starch in the flesh from depletion by the fungus. Further results demonstrated that CA induced reduction in mitochondrial membrane potential (Δψm), ROS accumulation, and cell apoptosis characterized by DNA fragmentation in F. solani. Moreover, CA facilitated decomposition of mitochondria-specific cardiolipin (CL) into its catabolites by the catalytic action of phospholipases. Altogether, the results revealed a specific antifungal mechanism of CA against F. solani, and suggest that CA holds promise as a preservative for postharvest preservation of sweet potato.
Assuntos
Fusarium , Ipomoea batatas , Antifúngicos/farmacologia , Ipomoea batatas/microbiologiaRESUMO
Root rot caused by Fusarium solani is one of the major postharvest diseases limiting sweet potato production. Here, antifungal activity and the action mode of perillaldehyde (PAE) against F. solani were investigated. A PAE concentration of 0.15 mL/L in air (mL/L air) markedly inhibited the mycelial growth, spore reproduction and spore viability of F. solani. A PAE vapor of 0.25 mL/L in air could control the F. solani development in sweet potatoes during storage for 9 days at 28 °C. Moreover, the results of a flow cytometer demonstrated that PAE drove an increase in cell membrane permeability, reduction of mitochondrial membrane potential (MMP) and accumulation of reactive oxygen species (ROS) in F. solani spores. Subsequently, a fluorescence microscopy assay demonstrated that PAE caused serious damage to the cell nuclei in F. solani by inducing chromatin condensation. Further, the spread plate method showed that the spore survival rate was negatively correlated with the level of ROS and nuclear damage, of which the results indicated that PAE-driven ROS accumulation plays a critical role in contributing to cell death in F. solani. In all, the results revealed a specific antifungal mechanism of PAE against F. solani, and suggest that PAE could be a useful fumigant for controlling the postharvest diseases of sweet potatoes.