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

• Published in: Aquaculture Vol. 604; p. 742482
• Main Authors: Ren, Jingqi, Liu, Jiaxi, Xiong, Dan, Wang, Xinyan, Zhang, Anying, Zhou, Hong
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 30.06.2025
• Subjects: aquaculture; autophagosomes; autophagy; CRISPR-Cas systems; Ctenopharyngodon idella; Edwardsiella piscicida; EseD; ferritin; Ferritinophagy; fish; Fish cell; genes; homeostasis; iron; lysosomes; macrophages; monocytes; NCOA4; Edwardsiella piscicida; Ferritinophagy; EseD; Fish cell; NCOA4
• DOI: 10.1016/j.aquaculture.2025.742482
• ISSN: 0044-8486

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.