Abstract
Edwardsiella ictaluri is a Gram-negative facultative intracellular fish pathogen causing enteric septicemia of catfish (ESC). While various secretion systems contribute to E. ictaluri virulence, the Type VI secretion system (T6SS) remains poorly understood. In this study, we constructed 13 E. ictaluri T6SS mutants using splicing by overlap extension PCR and characterized them, assessing their uptake and survival in channel catfish (Ictalurus punctatus) peritoneal macrophages, attachment and invasion in channel catfish ovary (CCO) cells, in vitro stress resistance, and virulence and efficacy in channel catfish. Among the mutants, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO exhibited reduced replication inside peritoneal macrophages. EiΔevpM, EiΔevpN, and EiΔevpO showed significantly decreased attachment to CCO cells, while EiΔevpN and EiΔevpO also displayed reduced invasion of CCO cells (p < 0.05). Overall, T6SS mutants demonstrated enhanced resistance to oxidative and nitrosative stress in the nutrient-rich medium compared to the minimal medium. However, EiΔevpA, EiΔevpH, EiΔevpM, EiΔevpN, and EiΔevpO were susceptible to oxidative stress in both nutrient-rich and minimal medium. In fish challenges, EiΔevpD, EiΔevpE, EiΔevpG, EiΔevpJ, and EiΔevpK exhibited attenuation and provided effective protection against E. ictaluri wild-type (EiWT) infection in catfish fingerlings. However, their attenuation and protective efficacy were lower in catfish fry. These findings shed light on the role of the T6SS in E. ictaluri pathogenesis, highlighting its significance in intracellular survival, host cell attachment and invasion, stress resistance, and virulence. The attenuated T6SS mutants hold promise as potential candidates for protective immunization strategies in catfish fingerlings.