Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Abstract

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.

Keywords: Aspergillus flavus; aflatoxin production; lysine succinylation; natural environments; quantification proteome.

Graphical abstract

Fig. 1

Phenotypes of high and low AF-production Aspergillus flavus strains.A, colony morphology and microscopic observation of AF-HA, AF-HB, AF-HC, AF-LA, and AF-LB strains cultured on plates at 37 °C for 3 days. B, growth assays of five strains on PDA plates. C, quantitative analysis of the conidial amounts of five strains. D, five strains inoculated on YPD plate at 37 °C for 7 days, the sclerotia were observed before or after washed by 75% ethanol. E, growth assays of five strains on YPD plates. F, quantitative analysis of the sclerotia amounts of five strains. G, the concentration of AFB₁ was assessed by LC–MS. The asterisks ∗∗∗ represents a significant difference level of p < 0.001. H, Western blot analysis of lysine succinylation in high and low AF-production A. flavus strains. AF, aflatoxin; AFB₁, aflatoxin B₁; PDA, potato dextrose agar; YPD, yeast peptone dextrose.

Fig. 2

Profiling of tandem mass tag (TMT)-based quantitative proteomics in high- and low-AF yielding Aspergillus flavus strains.A, workflow: experimental approach used to identify succinylated peptides. B, distribution of the lysine-succinylated peptides. C, histogram of the number of Ksuc sites per protein. D, Venn diagram showing the total numbers of Ksuc sites identified in high/low AF production strains. E, a representative MS/MS spectrum of a succinylated peptide from the VBS. F, Western blotting analysis of immunoprecipitated VBS. AF, aflatoxin; Ksuc, lysine succinylation; VBS, versicolorin B synthase.

Fig. 3

Tandem mass tag (TMT)–based quantitative proteomic analysis of lysine succinylation proteome.A, Volcano map showing the quantification of lysine succinylation sites in relation to peptide intensities. B, functional analysis of the succinylated proteins, the identified succinylated proteins enrichment of Gene Ontology (GO) categories for biological processes, molecular functions, and cellular components were performed using DAVID bioinformatics tools (p < 0.05). C, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment clustering showed by bubble chart. D, subcellular localization of identified succinylated proteins.

Fig. 4

Overview of the succinylated proteins involved in metabolism and aflatoxin biosynthesis pathways in Aspergillus flavus. The identified succinylated proteins were highlighted in red.

Fig. 5

vbs gene is involved in fungal vegetative growth and conidiation.A, colony morphology of WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains cultured on potato dextrose agar (PDA) and yeast extract with supplement (YES) plates at 37 °C for 3 days. B, growth assays of WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains. C, microscopic observation of asexual development. The conidiophores WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains were observed after constant light induction for 12 h. Bars represent 20 μm. D, conidial amounts of WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains. Conidia were extracted from each vegetative growth plate and counted by a microscope. E, quantitative RT–PCR (qRT–PCR) results showed that two regulatory genes for conidiation were regulated in WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C. β-actin was used as a reference. The asterisks ∗∗ represent a significant difference level of p < 0.01.

Fig. 6

Effects of vbs on sclerotia formation and aflatoxin (AF) biosynthesis in Aspergillus flavus.A, WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains were cultured on the yeast peptone dextrose (YPD) plate at 37 °C for 7 days, and the images were taken after the colony was washed with 75% ethanol to expose the sclerotia. B, the sclerotia amounts WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains. C, quantitative RT–PCR (qRT–PCR) results showed that two regulatory genes for sclerotia formation were regulated in WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C. β-actin was used as a reference. D, WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C strains were cultured on the yeast extract with supplement (YES) plate at 29 °C for 6 days, and TLC plates showed AF production extracted from aforementioned strains. E, the AFB₁ production WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C. F, qRT–PCR results showed that two genes for AF biosynthesis were regulated WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^C. β-actin was used as a reference. The asterisks ∗∗ represent a significant difference level of p < 0.01 and ∗ represent p < 0.05. AFB₁, aflatoxin B₁.

Fig. 7

Pathogenicity analysis of WT, Δvbs, Δvbs^K135R, Δvbs^K135A, and Δvbs^Cstrains.A, morphology of Aspergillus flavus on peanut after 5 days of inoculation. Mock means seed inoculated with sterile water as control. B, conidia production from infected seeds. C, TLC measurement of AFB₁ extracted from the infected seeds. D, quantification analysis of AFB₁ from the infected seeds. The asterisks ∗∗∗ represent a significant difference level of p < 0.001 and ∗∗ represent  p < 0.01. AFB₁, aflatoxin B₁.

Supplemental Figure S1

TLC and high-performance liquid chromatography analysis of aflatoxin production in the five different strains, which were cultured in PDB medium at 28 °C for 5 days.

Supplemental Figure S2

Reproducibility and accuracy of the quantitative proteomic analysis on.A, Pearson correlation coefficient for three repeated experiments. B, RSD (relative standard deviation) analysis of TMT labeling quantification proteomic.

Supplemental Figure S3

Analysis of Lys-succinylation.A, sequence properties of Lys-succinylation sites in high/low aflatoxin production strains. B, probable localization Lys-succinylation and all lysine in different secondary structures (α-helix, beta-strand, and coil) and protein surface.

Supplemental Figure S4

GO annotation information of identified proteins.A, classification of all differentially quantified proteins in GO terms of level 2. B, distribution of up-regulated proteins in GO terms of level 2. C, distribution of down-regulated proteins in GO terms of level 2.

Supplemental Figure S5

Analysis of the interaction network of identified succinylated proteins.

Supplemental Figure S6

Phylogenetic and structure analysis of VBS proteins.A, phylogenetic analysis of VBS proteins from Aspergillus members and other fungi. Bootstrap values were calculated using the neighbor-joining method with 1000 replicates A. flavus was underlined in red. B, schematic diagrams of VBS proteins and conserved domain was signed, respectively.

Supplemental Figure S7

Schematic for vbs deletion and complement.A, schematic for replacing the vbs gene in A. flavus by pyrg from Aspergillus fumigates. B, PCR diagnosis was carried out to confirm the deletion of genes and primers were listed in supplemental Table S1.

Supplemental Figure S8

Schematic for vbs^k135A/Rsite mutation.A, schematic for replacing the vbs gene in A. flavus by pyrg from Aspergillus fumigates and fusion vbsk^135A/R fragments. B, PCR diagnosis was carried out to confirm mutation of genes. Primers were listed in supplemental Table S1.

Supplemental Figure S9

Identification of site mutation strains.A, verification of point mutation vbs^k135A sequencing from WT and point mutation strains. B, verification of point mutation vbs^k135R sequencing.