Aspergillus flavus (A. flavus) is a ubiquitous saprophytic and pathogenic fungus that produces the aflatoxin carcinogen, and A. flavus can have tremendous economic and health impacts worldwide. Increasing evidence demonstrates that lysine succinylation plays an important regulatory role in metabolic processes in both bacterial and human cells. However, little is known about the extent and function of lysine succinylation in A. flavus. Here, we performed a global succinylome analysis of A. flavus using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 985 succinylation sites on 349 succinylated proteins were identified in this pathogen. Bioinformatics analysis revealed that the succinylated proteins were involved in various biological processes and were particularly enriched in the aflatoxin biosynthesis process. Site-specific mutagenesis and biochemical studies showed that lysine succinylation on the norsolorinic acid reductase NorA (AflE), a key enzyme in aflatoxins biosynthesis, can affect the production of sclerotia and aflatoxins biosynthesis in A. flavus. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and aflatoxins biosynthesis in A. flavus. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this pathogen.
In Aspergillus nidulans, the nitrogen metabolite repression (NMR) regulator NmrA plays a major role in regulating the activity of the GATA transcription factor AreA during nitrogen metabolism. However, the function of nmrA in A. flavus has not been previously studied. Here, we report the identification and functional analysis of nmrA in A. flavus. Our work showed that the amino acid sequences of NmrA are highly conserved among Aspergillus species and that A. flavus NmrA protein contains a canonical Rossmann fold motif. Deletion of nmrA slowed the growth of A. flavus but significantly increased conidiation and sclerotia production. Moreover, seed infection experiments indicated that nmrA is required for the invasive virulence of A. flavus. In addition, the ΔnmrA mutant showed increased sensitivity to rapamycin and methyl methanesulfonate, suggesting that nmrA could be responsive to target of rapamycin signaling and DNA damage. Furthermore, quantitative real-time reverse transcription polymerase chain reaction analysis suggested that nmrA might interact with other nitrogen regulatory and catabolic genes. Our study provides a better understanding of NMR and the nitrogen metabolism network in fungi.
Summary 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.
Aspergillus flavus is a pathogenic fungus that produces toxic and carcinogenic aflatoxins and is the causative agent of aflatoxicosis. A growing body of evidence indicates that reversible phosphorylation plays important roles in regulating diverse functions in this pathogen. However, only a few phosphoproteins of this fungus have been identified, which hampers our understanding of the roles of phosphorylation in A. flavus. So we performed a global and site-specific phosphoproteomic analysis of A. flavus. A total of 598 high-confidence phosphorylation sites were identified in 283 phosphoproteins. The identified phosphoproteins were involved in various biological processes, including signal transduction and aflatoxins biosynthesis. Five identified phosphoproteins associated with MAPK signal transduction and aflatoxins biosynthesis were validated by immunoblotting using phospho-specific antibodies. Further functional studies revealed that phosphorylation of the MAP kinase kinase kinase Ste11 affected aflatoxins biosynthesis in A. flavus. Our data represent the results of the first global survey of protein phosphorylation in A. flavus and reveal previously unappreciated roles for phosphorylation in the regulation of aflatoxins production. The generated dataset can serve as an important resource for the functional analysis of protein phosphorylation in A. flavus and facilitate the elucidation of phosphorylated signaling networks in this pathogen.
Aflatoxin is a toxic, carcinogenic mycotoxin primarily produced by Aspergillus parasiticus and Aspergillus flavus. Previous studies have predicted the existence of more than 20 genes in the gene cluster involved in aflatoxin biosynthesis. Among these genes, aflK encodes versicolorin B synthase, which converts versiconal to versicolorin B. Past research has investigated aflK in A. parasiticus, but few studies have characterized aflK in the animal, plant, and human pathogen A. flavus. To understand the potential role of aflK in A. flavus, its function was investigated here for the first time using gene replacement and gene complementation strategies. The aflK deletion‐mutant ΔaflK exhibited a significant decrease in sclerotial production and aflatoxin biosynthesis compared with wild‐type and the complementation strain ΔaflK::aflK. ΔaflK did not affect the ability of A. flavus to infect seeds, but downregulated aflatoxin production after seed infection. This is the first report of a relationship between aflK and sclerotial production in A. flavus, and our findings indicate that aflK regulates aflatoxin formation.
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