Aeromonas hydrophila effector Hcp1 triggers ferroptosis in teleost splenic macrophages via NCOA4-mediated ferritinophagy

The fish pathogenic bacterium Aeromonas hydrophila has been proved to trigger ferroptosis in grass carp splenic macrophages mainly via its secreted effectors, but the specific effectors involved remain unclear. In this study, recombinant proteins of several secretory effectors of A. hydrophila were prepared to detect their lipid peroxidation ability in grass carp splenic macrophages, showing that hemolysin coregulated protein 1 (Hcp1) could elevate lipid reactive oxygen species (ROS) production, and the role of Hcp1 in the bacterium-induced ferroptosis was reinforced by using a hcp1-deficient strain of A. hydrophila. Mechanistically, Hcp1 showed great potential to induce iron overload through a dual increase in cytosolic free Fe²⁺ based on ferritin degradation by nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy and ferroportin 1 (FPN1) ubiquitination. Moreover, Hcp1 also disturbed GSH/GPX4 antioxidant system by stimulating mitochondrial ROS production to deplete the cellular GSH, which was also driven by NCOA4-mediated ferritinophagy, ultimately impeding lipid ROS scavenging. These multiple effects of Hcp1 underlined its prominent role in A. hydrophila-induced ferroptosis in teleost. Collectively, Hcp1 is a potential effector to elicit ferroptosis via NCOA4-mediated ferritinophagy in teleost, providing a new insight into the pathogenic mechanism of A. hydrophila.