Edwardsiella piscicida effector EseD induces ferritinophagy to manipulate iron homeostasis and bacterial intracellular survival via nuclear receptor coactivator 4 (NCOA4) in fish cells

Edwardsiella piscicida has been reported to induce an autophagy-dependent iron disorder via secreted effectors for its intracellular survival in grass carp monocytes/macrophages. However, the effector involved in this event remains unknown. This study aimed to identify the effector and elucidate the mechanism of E. piscicida effector-manipulated iron homeostasis . By overexpressing several effectors of E. piscicida in fish cells, EseD, a critical component of the type III secretion system , was screened as a trigger for ferritin degradation and free iron accumulation. Furthermore, EseD displayed the ability to enhance intracellular survival of E. piscicida , implying the role of EseD-mediated iron disturbance in the bacterial intracellular survival. Mechanistically, EseD-induced ferritin degradation was found to be subject to autophagy. In support of this, colocalization analysis revealed that EseD facilitated ferritin to target the autophagosomes and lysosomes , reinforcing the role of autophagy in EseD-mediated iron disorders. These findings prompted us to assess whether EseD induced ferritinophagy, a process involving selective autophagy of ferritin . In this case, NCOA4 (nuclear receptor coactivator 4, a selective cargo receptor binding ferritin for ferritinophagy) gene was knocked out by CRISPR/Cas9 in EPC cells. Results showed that NCOA4 deficiency interfered with EseD-caused ferritin degradation, increased intracellular free iron and ferritin-colocalized with autophagosomes in cells, suggesting the involvement of NCOA4-mediated ferritinophagy in EseD-disturbed iron homeostasis . Moreover, intracellular E. piscicida survival was attenuated in both absence and presence of EseD overexpression in NCOA4 deletion cells, highlighting the role of ferritinophagy in the intracellular growth of E. piscicida . In summary, this study implied that E. piscicida EseD elicited NCOA4-mediated ferritinophagy leading to iron disorder in fish cells and bacterial survival , providing a new insight into the interaction between E. piscicida and host cells. • E. piscicida effector EseD was proved to disturb iron homeostasis in fish cells. • EseD-caused iron disorder was subject to autophagic pathway. • Ferritinophagy via NCOA4 was required for EseD-disturbed iron homeostasis. • NCOA4-mediated ferritinophagy manipulated intracellular survival of E. piscicida .