Proteome-Wide Profiling of Lysine 2-Hydroxyisobutyrylation in Vibrio alginolyticus and Preliminary Association of CobQ with Global Khib Levels

Abstract

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.