Phosphoenolpyruvate Carboxykinase Controls Edwardsiella tarda Virulence via Oxaloacetate

Simple SummaryEdwardsiella tardais a bacterium that causes severe disease in fish, leading to major economic losses for aquaculture worldwide. Understanding how this bacterium causes infection is essential for developing effective prevention strategies. In this study, we investigated a key bacterial enzyme called phosphoenolpyruvate carboxykinase (PEPCK), which helps control the bacterium’s energy metabolism. When we removed the gene responsible for producing this enzyme, the bacteria became less harmful to fish, grew faster, and showed increased movement. Importantly, this modified bacterium triggered a strong immune response in fish and protected them against subsequent infection with the deadly form of the bacteria. Fish that received the modified bacteria survived at much higher rates compared to unprotected fish. These findings reveal that this enzyme plays a critical role in controlling bacterial disease-causing ability through its effects on cellular metabolism. The modified bacteria show promise as a safe and effective vaccine candidate to protect farmed fish againstEdwardsiellainfections, potentially reducing disease outbreaks and economic losses in aquaculture while decreasing the need for antibiotic treatments.Understanding the metabolic basis of bacterial virulence is essential for developing anti-infective strategies. Phosphoenolpyruvate carboxykinase (PEPCK), encoded bypckA, is a key enzyme at the metabolic junction between the pyruvate cycle and the TCA cycle, but its role in virulence regulation remains largely unexplored. In this study, we generated apckAdeletion mutant,ΔpckA, inEdwardsiella tardaand investigated its impact on virulence, metabolism, and vaccine potential. TheΔpckAmutant exhibited increased antibiotic sensitivity, faster growth, reduced autoaggregation, and significantly attenuated virulence in a tilapia infection model. Quantitative proteomics revealed global downregulation of type III and type VI secretion systems (T3SS and T6SS) and upregulation of flagellar proteins. Mechanistically, accumulation of oxaloacetate (OAA) in the mutant suppressed T3SS/T6SS expression without affecting flagellar motility. Importantly,ΔpckAinduced a robust immune response in zebrafish and conferred strong protection against lethal challenge, with relative percent survival values of 64% and 81.82% in zebrafish and tilapia, respectively. Taken together, our findings identify PEPCK as a critical metabolic regulator ofE. tardavirulence and highlightΔpckAas a promising live attenuated vaccine candidate.