Vibrio alginolyticus is a Gram-negative pathogen of both marine animals and humans, resulting in significant losses for the aquaculture industry. Emerging evidence indicates that V. alginolyticus manipulates cell death for its pathogenicity, but the underlying molecular mechanisms remain unclear. Here, a gene designated vopS in V. alginolyticus HY9901 was identified, which was predicted to encode the T3SS effector protein. To determine whether VopS contributes to the pathogenesis of V. alginolyticus, the ΔvopS mutant strain was constructed and phenotypically characterized. The deletion of VopS not only reduced the ability to secrete extracellular proteases and virulence but also affected the expression of the T3SS genes. Furthermore, VopS was cytotoxic and induced apoptosis, as confirmed by elevated LDH and the activation of caspase-3. Metabolomic analysis revealed considerable metabolomic disruptions upon V. alginolyticus infection. The VopS effector induced host cell ferroptosis by promoting the synthesis of adrenic acid, depleting cellular glutathione, and subsequently increasing the accumulation of ferrous (Fe²⁺). Taken together, our findings provide that the VopS effector is an essential virulence factor of V. alginolyticus, which can lead to ferroptosis.

Vibrio alginolyticus is a common pathogenic bacterium and can cause diseases in aquaculture animals. Lysine lactylation (Kla) is a novel post-translational modification (PTM) that has been confirmed to play critical roles in key biological processes. However, the modification landscape and functions of Kla in V. alginolyticus remain unclear. In this study, lactylation modification profiles of the bacterial pathogen V. alginolyticus were first systematically characterized; a total of 9308 lactylation sites on 2155 proteins were successfully identified. The lactylation of cAMP receptor protein (CRP) and triosephosphate isomerase (TPI) was verified by Co-immunoprecipitation (Co-IP) and Western blot to validate the lactylome data. Bioinformatic analysis of the Kla sites revealed 32 conserved sequence motifs surrounding the modified residues. Kla proteins were mainly involved in central metabolic pathways, including glycolysis/gluconeogenesis and ribosome biogen regulators were found to contain Kla modification sites. To investigate crosstalk among lysine acylations in V. alginolyticus, we integrated Kla, lysine acetylation (Kac), and lysine succinylation (Ksuc) profiles and identified 337 co-modified proteins and 5 co-modified sites. Additionally, phylogenetic analysis of Vibrio alginolyticus CobQ based on protein sequence alignment revealed no homology to the known delactylase CobB. Combined in vitro and in vivo functional validation identified VaCobQ as a candidate delactylase with potential NAD⁺-independent activity. This study establishes a lysine lactylation landscape in V. alginolyticus, providing a resource for exploring Kla functions in bacterial metabolism and its possible connections to virulence.

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.