First Succinylome Profiling of Vibrio alginolyticus Reveals Key Role of Lysine Succinylation in Cellular Metabolism and Virulence

Abstract

Recent studies have shown that a key strategy of many pathogens is to use post-translational modification (PTMs) to modulate host factors critical for infection. Lysine succinylation (Ksuc) is a major PTM widespread in prokaryotic and eukaryotic cells, and is associated with the regulation of numerous important cellular processes. Vibrio alginolyticus is a common pathogen that causes serious disease problems in aquaculture. Here we used the affinity enrichment method with LC-MS/MS to report the first identification of 2082 lysine succinylation sites on 671 proteins in V. alginolyticus, and compared this with the lysine acetylation of V. alginolyticus in our previous work. The Ksuc modification of SodB and PEPCK proteins were further validated by Co-immunoprecipitation combined with Western blotting. Bioinformatics analysis showed that the identified lysine succinylated proteins are involved in various biological processes and central metabolism pathways. Moreover, a total of 1,005 (25.4%) succinyl sites on 502 (37.3%) proteins were also found to be acetylated, which indicated that an extensive crosstalk between acetylation and succinylation in V. alginolyticus occurs, especially in three central metabolic pathways: glycolysis/gluconeogenesis, TCA cycle, and pyruvate metabolism. Furthermore, we found at least 50 (7.45%) succinylated virulence factors, including LuxS, Tdh, SodB, PEPCK, ClpP, and the Sec system to play an important role in bacterial virulence. Taken together, this systematic analysis provides a basis for further study on the pathophysiological role of lysine succinylation in V. alginolyticus and provides targets for the development of attenuated vaccines.

Keywords: Vibrio alginolyticus; acetylation; crosstalk; lysine succinylation; virulence factors.

Figure 1

Profile of V. alginolyticus lysine succinylation proteome. (A) Distributions of mass errors for lysine succinylated peptides. (B) Distribution of lysine succinylated peptides based on their length. (C) The pie chart shows the distribution of succinylation sites in each protein.

Figure 2

Gene ontology functional classification and KEGG pathway analysis of the identified succinylated proteins. (A) Molecular function. (B) Biological processes. (C) Cell components. (D) KEGG pathway analysis.

Figure 3

Functional annotation of the lysine succinylome in V. alginolyticus. (A) The COG analysis and (B) domains enrichment analysis of the succinylated proteins.

Figure 4

Motif analysis of lysine succinylation sites. (A) Sequence logos of motifs (P-value <0.000001) identified by MoMo software. (B) Numbers of each identified motifs.

Figure 5

Validation of SodB and PEPCK lysine-succinylated proteins in V. alginolyticus using Co-Immunoprecipitation and Western blotting. SodB and PEPCK proteins were enriched by Co-IP with specific antibodies, followed by Western blotting with SodB and PEPCK proteins specific antibodies (above), and Western blotting with anti-lysine succinylation antibodies (below).

Figure 6

Comparison of succinylated and acetylated proteins in V. alginolyticus. (A) Overlap between succinylated and acetylated proteins in V. alginolyticus. (B–D) KEGG pathway enrichment analysis of the overlapped proteins, specific succinylated modified proteins, and specific acetylated modified proteins.

Figure 7

Comparison of succinylation and acetylation peptides in V. alginolyticus Overlap between succinylated and acetylated peptides in V. alginolyticus, and motif analysis.

Figure 8

Key enzymes with succinylation and acetylation modification in glycolysis/gluconeogenesis, TCA cycle, and pyruvate metabolism pathways.

Figure 9

The PPI network of succinylated virulence factors in V. alginolyticus. The size of the circle represents the number of Ksuc sites.