Global Insight into Lysine Acetylation Events and Their Links to Biological Aspects in Beauveria bassiana, a Fungal Insect Pathogen

Abstract

Lysine acetylation (Kac) events in filamentous fungi are poorly explored. Here we show a lysine acetylome generated by LC-MS/MS analysis of immunoaffinity-based Kac peptides from normal hyphal cells of Beauveria bassiana, a fungal entomopathogen. The acetylome comprised 283 Kac proteins and 464 Kac sites. These proteins were enriched to eight molecular functions, 20 cellular components, 27 biological processes, 20 KEGG pathways and 12 subcellular localizations. All Kac sites were characterized as six Kac motifs, including a novel motif (KacW) for 26 Kac sites of 17 unknown proteins. Many Kac sites were predicted to be multifunctional, largely expanding the fungal Kac events. Biological importance of identified Kac sites was confirmed through functional analysis of Kac sites on Pmt1 and Pmt4, two O-mannosyltransferases. Singular site mutations (K88R and K482R) of Pmt1 resulted in impaired conidiation, attenuated virulence and decreased tolerance to oxidation and cell wall perturbation. These defects were close to or more severe than those caused by the deletion of pmt1. The Pmt4 K360R mutation facilitated colony growth under normal and stressful conditions and enhanced the fungal virulence. Our findings provide the first insight into the Kac events of B. bassiana and their links to the fungal potential against insect pests.

Figure 1. Outline of B. bassiana acetylome.

(A) Workflow for creation of the acetylome through digestion of proteins with trypsin and enrichment of acetylated peptides, nano-LC/MS/MS analysis and bioinformatic annotation. (B,C) Distributions of mass errors and lengths for all identified Kac peptides. (D) Percentages of Kac proteins vectoring 1, 2, 3, 4, and ≥5 Kac sites, respectively. (E) Mean probabilities of lysine acetylation predicted for different types of protein secondary structures (α-helix, β-strand and coil). (F) Predicted surface accessibility of acetylated sites. The marked p values denote a significance of the non-paired Wilcox test for the predicted values in (E,F).

Figure 2. Features of B. bassiana acetylome.

(A–D) Fold enrichment and Fisher’s p values for the Kac proteins enriched to molecular functions, cellular components, biological processes and KEGG pathways through GO and KEGG analyses, respectively.

Figure 3. Kac motifs identified from the B. bassiana acetylome and their preferences to other amino acid residues.

(A) Logos of six Kac motifs predicted with the WebLogo software at http://weblogo.berkeley.edu. The height of each amino acid indicates the level of conservation at that position. Each logo is followed by the outlined sequence of each motif, the motif score, the foreground counts of matches (F_match)/identified Kac peptides (F_size), the background counts of matches (B_match)/all the identified peptides (B_size) in the used database, and the fold increase, respectively. Fold increase = (F_size/F_match)/(B_size/B_match). (B) Enriched preferences of Kac sites to amino acid residues at particular positions. Red: significantly preferred residues. Green: not enriched around Kac sites.

Figure 4. Interaction networks of Kac proteins in B.bassiana.

Protein-to-protein interactions revealed by Cytoscape software occur among the top five clusters associated with ribosome (red), glycolysis/gluconeogenesis (pink), histone (blue), proteasome (dark blue), and protein processing in endoplasmic reticulum (green) respectively.

Figure 5. LC-MS/MS spectra for identification of Kac sites from Pmt1 and Pmt4 (O-mannosyltransferases) in the B. bassiana acetylome.

Relative abundance (left Y-axis) and peak density (right Y-axis) of accumulated ions over the marked m/z range indicate that the acetylated residues are K88 (A) and K482 (B) on Pmt1 and K360 (C) on Pmt4.

Figure 6. Phenotypes of B. bassiana altered by pmt1 or pmt4 deletion and singular Kac site mutations.

(A) Diameters of fungal colonies after 7 days of incubation at 25 °C in rich SDAY, 1/4 SDAY and minimal CZA alone or in 1/4 SDAY supplemented with NaCl (0.8 M), H₂O₂ (4 mM) and CGR (Congo red 80 mg/ml) respectively. Each colony was initiated with 1 μl of a 10⁶ conidia/ml suspension. (B) Conidial yields quantified daily from the SDAY cultures over the days of incubation at 25 °C in a light/dark cycle of 12:12 h. All cultures were initiated by spreading 100 μl of a 10⁷ conidia/ml suspension per plate. (C,D) LT₅₀ (no. days) for fungal virulence to G. mellonella larvae via cuticular penetration (immersed) and cuticle-bypassing infection (injected), respectively. Asterisked bars differ significantly from those unmarked in each group of bars within each chart (Tukey’s HSD, p < 0.05). Error bars: SD from three replicates.