Vibrio harveyi, a pathogenic vibrio, is ubiquitous and the most prevalent disease infecting tropical and subtropical mariculture animals in marine and estuarine environments. It presents a major risk to mariculture companies worldwide and can cause serious disease problems in aquaculture. Recent studies have shown that various pathogens employ post-translational modifications (PTMs) to regulate cellular processes. One of the major PTMs is lysine succinylation, which is widespread in eukaryotic and prokaryotic cells. Many basic biological functions of bacteria are associated with the regulation of lysine (K) succinylation (Ksuc). However, little is known about the role of lysine succinylation in V. harveyi pathogenesis. Here, we performed LC-MS/MS analysis of 1271 proteins from V. harveyi to identify 4252 Ksuc modification sites. The modification of S-ribosylhomocysteine lyase (LuxS) and transcription elongation factor GreA proteins by Ksuc was confirmed through immunoprecipitation combined with Western blot, further validating our proteomics results. Bioinformatics study revealed that the identified Ksuc proteins play roles in multiple cellular processes and vital metabolic pathways, including LuxS, biofilm exopolysaccharide biosynthesis protein EpsG, and the general secretory system (Sec systems), and are proteins that regulate bacterial virulence. Generally, this scientific study serves as the basis for additional research on the pathogenic nature of Ksuc in V. harveyi and reveals potential targets that would accelerate the manufacturing of attenuated vaccines.

Vibrio alginolyticus is an important opportunistic pathogen in marine aquaculture, causing vibriosis in a wide range of aquatic animals. Lysine 2-hydroxyisobutyrylation (Khib) is an emerging lysine acylation modification involved in regulating metabolism, stress responses, and pathogenicity in microorganisms. However, the global Khib modification profile and its potential biological functions in V. alginolyticus have remained completely unexplored. This study aims to characterize the Khib modification profile of V. alginolyticus and to preliminarily evaluate the role of CobQ as a candidate regulator of Khib homeostasis. A Khib-modified proteome of V. alginolyticus was constructed using affinity enrichment combined with LC-MS/MS-based modified proteomics. Bioinformatics analyses (GO, COG, KEGG, subcellular localization, motif, and protein–protein interaction) explored the functions of Khib-modified proteins. Selected proteins were analyzed by immunoprecipitation combined with Western blot. A cobQ deletion mutant strain was constructed to assess the association between cobQ deletion and global Khib levels, and to evaluate selected physiological traits. A total of 15,195 Khib sites were identified in 2848 proteins, indicating widespread distribution of Khib modification. Khib-modified proteins were mainly associated with metabolic processes, genetic information processing, signal transduction, membrane biogenesis, and secretion-related pathways. Motif analysis revealed conserved amino acid patterns surrounding Khib sites. Immunoprecipitation combined with Western blot confirmed the Khib modification of TpiA and Ndk at the protein level, but did not validate the specific LC-MS/MS sites. Deletion of cobQ was associated with increased global Khib levels, suggesting that CobQ may be associated with Khib modification in vivo. In vitro assays showed no statistically significant de-Khib activity of CobQ, although a trend toward lower Khib levels was observed. Phenotypic assays showed that cobQ deletion did not significantly affect growth or extracellular protease activity, but reduced swimming motility at low agar concentrations. This study provides a comprehensive catalog of putative Khib-modified proteins of V. alginolyticus. Our findings suggest that CobQ may be associated with global Khib levels, but its direct enzymatic activity and physiological substrates remain to be established. These findings provide a foundational resource for understanding Khib-mediated regulation in V. alginolyticus and identify candidate targets for future mechanistic studies.

Acetylation modification has become one of the most popular topics in protein post-translational modification (PTM) research and plays an important role in bacterial virulence. A previous study indicated that the virulence-associated caseinolytic protease proteolytic subunit (ClpP) is acetylated at the K165 site in Vibrio alginolyticus strain HY9901, but its regulation regarding the virulence of V. alginolyticus is still unknown. We further confirmed that ClpP undergoes lysine acetylation (Kace) modification by immunoprecipitation and Western blot analysis and constructed the complementation strain (C-clpP) and site-directed mutagenesis strains including K165Q and K165R. The K165R strain significantly increased biofilm formation at 36 h of incubation, and K165Q significantly decreased biofilm formation at 24 h of incubation. However, the acetylation modification of ClpP did not affect the extracellular protease (ECPase) activity. In addition, we found that the virulence of K165Q was significantly reduced in zebrafish by in vivo injection. To further study the effect of lysine acetylation on the pathogenicity of V. alginolyticus, GS cells were infected with four strains, namely HY9901, C-clpP, K165Q and K165R. This indicated that the effect of the K165Q strain on cytotoxicity was significantly reduced compared with the wild-type strain, while K165R showed similar levels to the wild-type strain. In summary, the results of this study indicate that the Kace of ClpP is involved in the regulation of the virulence of V. alginolyticus.