Edwardsiella piscicida effector VgrG contributes to NCOA4-dependent ferritinophagy as a bacterial survival strategy in fish.

Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal injury and hemorrhagic sepsis in marine and freshwater animals. VgrG (valine-glycine repeat protein G) has been identified as a crucial virulence factor in the type VI secretion system (T6SS) of the bacterium, but its role and mechanism involved in E. piscicida-host interactions remain unclear. In this study, we found that the wild-type E. piscicida strain markedly induced host cell ferritin degradation and elevated intracellular Fe²⁺ levels, but a vgrG deletion mutant (ΔvgrG) strain failed to induce similar effects in fish cells, indicating the role of VgrG in the regulation of iron metabolism. However, in the NCOA4 (nuclear receptor coactivator 4, a selective cargo receptor that binds ferritin)-knockout fish cells, VgrG did not alter ferritin protein expression and E. piscicida-promoted intracellular Fe²⁺ levels, suggesting that VgrG caused iron storage disorders via NCOA4-dependent ferritinophagy. In support of this notion, VgrG overexpression was found to facilitate the co-localization of ferritin with autophagosomes and lysosomes, and also drove the interaction of NCOA4 with lysosomes, strengthening the involvement of this effector in mediating NCOA4-dependent ferritinophagy. Notably, in NCOA4-knockout cells, the inhibitory effect of VgrG on the intracellular growth of E. piscicida was further magnified, particularly highlighting the role of VgrG-induced ferritinophagy in augmenting bacterial intracellular survival. Moreover, zebrafish infected with the ΔvgrG strain had a much higher survival rate than those infected with the wild-type strain. In summary, our data revealed a new function of VgrG in maintaining the persistence of E. piscicida within host cells, which advanced our understanding of how intracellular pathogens target ferritinophagy to evade the host's immune surveillance. Moreover, this work also supported the potential for developing defense strategies against E. piscicida based on the VgrG-induced ferritinophagy response in fish.