Holistic analysis of lysine acetylation in aquaculture pathogenic bacteria Vibrio alginolyticus under bile salt stress

Abstract

Lysine acetylation modification is a dynamic and reversible post-translational modification, which plays an important role in the metabolism and pathogenicity of pathogenic bacteria. Vibrio alginolyticus is a common pathogenic bacterium in aquaculture, and bile salt can trigger the expression of bacterial virulence. However, little is known about the function of lysine acetylation in V. alginolyticus under bile salt stress. In this study, 1,315 acetylated peptides on 689 proteins were identified in V. alginolyticus under bile salt stress by acetyl-lysine antibody enrichment and high-resolution mass spectrometry. Bioinformatics analysis found that the peptides motif ^****A^*Kac^**** and ^*******Kac^****A^* were highly conserved, and protein lysine acetylation was involved in regulating various cellular biological processes and maintaining the normal life activities of bacteria, such as ribosome, aminoacyl-tRNA biosynthesis, fatty acid metabolism, two-component system, and bacterial secretion system. Further, 22 acetylated proteins were also found to be related to the virulence of V. alginolyticus under bile salt stress through secretion system, chemotaxis and motility, and adherence. Finally, comparing un-treated and treated with bile salt stress lysine acetylated proteins, it was found that there were 240 overlapping proteins, and found amino sugar and nucleotide sugar metabolism, beta-Lactam resistance, fatty acid degradation, carbon metabolism, and microbial metabolism in diverse environments pathways were significantly enriched in bile salt stress alone. In conclusion, this study is a holistic analysis of lysine acetylation in V. alginolyticus under bile salt stress, especially many virulence factors have also acetylated.

Keywords: Vibrio alginolyticus; acetylome; bile salt; post-translational modification; virulence.

Figure 1

Outline of acetylome under bile salt stress in V. alginolyticus. (A) Site coverage of acetylated proteins, and (B) Distribution of acetylated peptide lengths.

Figure 2

Analysis of motifs of lysine acetylated-peptides under bile salt stress in V. alginolyticus. (A) The sequence logos of the acetylation motifs identified by MoMo software. These motifs consist of 14 residues (seven amino acids upstream and seven amino acids downstream) surrounding the acetylated lysine, and (B) Number of acetylated peptides contained in the identified motif.

Figure 3

GO and KEGG enrichment analysis of lysine acetylated proteins under bile salt stress in V. alginolyticus. (A) biological process, (B) molecular function, (C) cellular component, and (D) KEGG pathway.

Figure 4

Functional annotation of the lysine acetylated proteins under bile salt stress in V. alginolyticus. (A) The COG function classification analysis and (B) subcellular localization analysis of the acetylated proteins.

Figure 5

Protein-protein interaction networks of acetylated proteins under bile salt stress in V. alginolyticus. PPI networks of acetylated proteins were analyzed using the STRING combine with Cytoscape software. (A) ribosomes, (B) two-component system, (C) aminoacyl-tRNA biosynthesis, (D) fatty acid metabolism, and (E) bacterial secretion system.

Figure 6

Validation of OmpN (A), OmpR (B), and GrpE (C) by Co-IP and Western blotting. OmpN, OmpR, and GrpE proteins were captured by specific antibodies (anti-OmpN, anti-OmpR, and anti-GrpE), and validation by Western blotting with anti-OmpN, anti-OmpR and anti-GrpE (above), and anti-lysine acetylation antibodies (below).

Figure 7

Comparison of un-treated and treated with bile salt stress lysine acetylated proteins in V. alginolyticus. (A) Overlap between un-treated and treated bile salt stress lysine acetylated proteins in V. alginolyticus, (B) GO enrichment analysis, and (C) KEGG pathway enrichment analysis of the overlapped acetylated proteins. (D, E) KEGG pathway enrichment analysis of treated and un-treated with bile salt stress lysine acetylation protein.