Lysine Succinylation Contributes to Aflatoxin Production and Pathogenicity in Aspergillus flavus

Abstract

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.

Fig. 1.

Profiling lysine propionylation in A. flavus NRRL 3357. A, Morphological phenotypes of A. flavus on different media. 1: standard YES media, 2: improved media, in which sucrose was replaced by 18.75 g/L sodium succinate, 3: improved media, in which sucrose was replaced by 37.5 g/L sodium succinate, 4: improved media, in which sucrose was replaced by 75 g/L sodium succinate, 5: improved media, in which sucrose was replaced by 150 g/L sodium succinate. B, Quantitative analysis of spore grown on different media (p value < 0.01). C, Thin-layer chromatography analysis of aflatoxin production of A. flavus grown on different media. D, Ponceau staining of protein lysates from A. flavus grown on different media and Western blotting analysis of lysine succinylation in A. flavus grown on different media. E, Workflow for lysine succinylome analysis of A. flavus. F, A representative MS/MS spectrum of a succinylated peptide from the norsolorinic acid reductase NorA (AflE).

Fig. 2.

Enrichment analysis of succinylated proteins and bioinformatics analysis of succinylation sites. A, Histogram representations of the enrichment of identified succinylated proteins for biological processes, molecular functions, cellular components and KEGG pathways. The enrichment of GO categories, pathway and domain were performed using DAVID bioinformatics tools (p < 0.05). B, Motif-X analysis of the succinylated sites. The motifs with significance of p < 0.000001 are shown. C, Heat map showing sequence motifs of lysine-succinylated sites. The intensity map shows the relative abundance for ± 6 amino acids from the lysine-succinylated site. The colors in the intensity map represent the log₁₀ of the ratio of frequencies within succinyl-13-mers versus non-succinyl-13-mers (red shows enrichment, yellow shows depletion).

Fig. 3.

Protein interaction networks of all identified succinylated proteins. The interaction network was visualized with Cytoscape.

Fig. 4.

Central metabolism and aflatoxins biosynthesis pathways in A. flavus. Succinylated proteins were highlighted in red. ACC: acetyl-CoA carboxylase. PksA: noranthrone synthase, AflC/PksA/PksL1/polyketide synthase. Fas1: AflB/Fas-1/fatty acid synthase beta subunit. Fas2: AflA/Fas-2/HexA/fatty acid synthase alpha subunit. HypC: noranthrone monooxygenase, AflCa/HypC/hypothetical protein. AflD: Nor-1/norsolorinic acid ketoreductase. AflF: NorB/dehydrogenase. AvnA: averantin hydroxylase, AflG/AvnA/Ord-1/cytochrome P450 monooxygenase. AdhA: 5′-hydroxyaverantin dehydrogenase, AflH/AdhA/short chain alcohol dehydrogenase. AvfA: Oxidase, AflI/AvfA/cytochrome P450 monooxygenase. VrdA: versiconal hemiacetal acetate reductase/aryl-alcohol dehydrogenase. EstA: versiconal hemiacetal acetate esterase, AflJ/EstA/esterase. VBS: 5′-oxoaverantin cyclase/versicolorin B synthase, AflK/Vbs/VERB synthase. AflN: VerA/monooxygenase. AflM: Ver-1/dehydrogenase/ketoreductase. OmtB: DmtA, demethylsterigmatocystin 6-O-methyltransferase, AflO/OmtB/DmtA/O-methyltransferase B. OmtA: sterigmatocystin 8-O-methyltransferase, AflP/OmtA/Omt-1/O-methyltransferase A. OrdA: aflatoxin B synthase, AflQ/OrdA/Ord-1/oxidoreductase/cytochrome P450 monooxigenase. AflE: NorA/Aad/Adh-2/NOR reductase/dehydrogenase. ACAT: acetyl-CoA acetyltransferase. PDC: pyruvate dehydrogenase complex. LDH: lactate dehydrogenase. ACL: ATP citrate lyase. HMG-CoA: 3-hydroxy-3-methylglutaryl-CoA. ACH: acetyl-coA hydrolase. 6PGL: 6-Phosphogluconolactonase. 3PG: 3-phosphoglycerate. 2PG: 2-phosphoglycerate. PGM: phosphoglycerate mutase. PK: pyruvate kinase. PEP: phosphoenolpyruvate. F6P: β-d-Fructose 6-phosphate. PFK: 6-phosphofructokinase. F1,6BP: β-d-Fructose 1,6-bisphosphate. ALDO: fructose-bisphosphate aldolase. GADP: d-glyceraldehyde 3-phosphate. DHAP: dihydroxyacetone phosphate. GAPDH: glyceraldehyde phosphate dehydrogenase. 1,3BPG: d-1,3-bisphosphoglycerate. G6P: glucose-6-phosphate. G: glucose. G1P: glucose-1-phosphate. PgmA: phosphoglucomutase. PdcA: pyruvate decarboxylase. AH: aconitate hydratase. Gnd1: 6-phosphogluconate dehydrogenase. IDH: isocitrate dehydrogenase. TktA: transketolase. PC: pyruvate carboxylase. OAA: oxaloacetate. GPI: glucose-6-phosphate isomerase. MDH: malate dehydrogenase. PgkA: phosphoglycerate kinase. UTP-G1PU: UTP-glucose-1-phosphate uridylyltransferase. TPI: triose-phosphate isomerase. CIT1: citrate synthase. ADH: alcohol dehydrogenases. SDH1: succinate dehydrogenase.

Fig. 5.

Sclerotial characterization of different A. flavus strains. A, Morphological phenotypes of sclerotia in WT, ΔaflE, K370R, K370A and ΔaflE::aflE strains on WKM media. B, Quantification analysis of sclerotia. Sclerotial production was counted from three replicates of WKM plates in (A). The corresponding p value < 0.01 was considered statistically significant.

Fig. 6.

Aflatoxin production in different A. flavus strains. A, Thin layer chromatography analysis of aflatoxin B₁ in WT, ΔaflE, K370R, K370A and ΔaflE::aflE strains. B, Quantification analysis of aflatoxin B₁ according to the result of thin layer chromatography. The corresponding p value < 0.01 was considered statistically significant. C, HPLC analysis of aflatoxins production in WT, ΔaflE, K370R, K370A and ΔaflE::aflE strains.

Fig. 7.

Host colonization of different A. flavus strains. A, Phenotypic characterization of WT, ΔaflE, K370R, K370A and ΔaflE::aflE strains on peanut cotyledons for 4 d. B, Quantification of conidia. Conidial production was counted from three replicates of the A. flavus strains in (A). The corresponding p value < 0.01 was considered statistically significant. C, Thin layer chromatography analysis of aflatoxin B₁ collected from infected peanut cotyledons. D, Quantification of AFB₁. AFB₁ production was counted from three replicates of the A. flavus strains in (C). The corresponding p value < 0.01 was considered statistically significant.

Fig. 8.

Schematic summary of the proposed model of the role of lysine succinylation in aflatoxins biosynthesis and sclerotial formation.