Background Pseudomonas plecoglossicida is a marine bacterial pathogen that poses a serious threat to the aquaculture industry. However, the mechanisms by which changes in environmental ion concentrations affect its virulence remain poorly understood.

Objective This study aims to systematically evaluate the effects of the metal chelator EGTA on the virulence of P. plecoglossicida.

Methods Bacterial growth curves combined with transmission electron microscopy were used to assess the effects of EGTA on growth status and cellular morphological integrity of P. plecoglossicida. A lactate dehydrogenase (LDH) release assay was performed to evaluate differences in cytotoxicity between culture supernatants of EGTA-treated and untreated bacteria toward Epithelioma papulosum cyprini (EPC) cells. Comparative secretomics was employed to characterize the dynamic changes in the secretome profiles of bacterial culture supernatants before and after EGTA treatment. Differentially secreted proteins were ectopically expressed in EPC cells, and potential virulence factors were screened using the LDH release assay. In addition, western blotting (WB) and reverse transcription quantitative PCR (RT-qPCR) were used to determine changes in the expression of the type III secretion system (T3SS) effector exoU and its upstream regulatory gene exsA at the protein and transcriptional levels, respectively.

Results The results showed that 5 mmol/L EGTA significantly enhanced the in vitro cytotoxicity of P. plecoglossicida toward EPC cells without altering bacterial cellular morphology or integrity. Comparative secretomic analysis revealed that EGTA treatment markedly altered the bacterial secretome, with 204 and 226 differentially secreted proteins identified at 12 h and 24 h, respectively, predominantly showing upregulation. Ectopic expression in EPC cells confirmed that several differentially expressed proteins, including the core components of the type VI secretion system (T6SS) (TssJ and TssF), a specific amidase, and a histidine kinase, could independently induce significant host cell lysis. Unlike the classical mechanism in Gram-negative pathogens where EGTA activates the T3SS, neither the secretion of the T3SS effector protein ExoU nor the transcription levels of the exoU gene and its upstream regulator exsA showed significant changes under EGTA stress.

Conclusion This study is the first to reveal the effects of EGTA on the secretion of virulence proteins in P. plecoglossicida, laying a solid foundation for further elucidation of its pathogenic mechanisms.