Aeromonas hydrophila induces mitophagy for intracellular survival through its secreted effectors in the splenic macrophages of grass carp (Ctenopharyngodon idella).

Aeromonas hydrophila, a Gram-negative bacterium ubiquitously distributes in aquatic environments, represents a prevalent pathogenic threat to aquatic animals. The present study investigated how A. hydrophila induced mitophagy to promote its own survival in the splenic macrophages of grass carp (Ctenopharyngodon idella). Our results revealed that A. hydrophila impaired mitochondrial integrity by reducing mitochondrial membrane potential and ATP levels, as well as increasing mitochondrial ROS (mtROS) production. Meanwhile, this bacterium decreased the expression of the TOM20, a mitochondrial outer membrane protein, accompanied by an increased colocalization of the autophagy marker LC3 with TOM20, as well as an enhanced fusion of mitochondria with lysosomes. These effects were reversed by the mitophagy inhibitor Mdivi-1, confirming the occurrence of A. hydrophila-induced mitophagy. Subsequently, we found that both live bacteria and their culture supernatants but not heat-inactivated supernatants triggered the mitophagy responses. Mechanistical evidence of A. hydrophila-induced mitophagy by the PINK1-Parkin pathway included the upregulation of PINK1/Parkin expression and the blockade of TOM20-LC3 colocalization by Oroxylin A, which specifically dampens the PINK1-Parkin pathway. Furthermore, the physiological significance of A. hydrophila-induced mitophagy was emphasized in the splenic macrophages where pretreatment with Mdivi-1 was found to significantly reduce the intracellular load of A. hydrophila. In contrast, the mitophagy inducer m-chlorophenylhydrazone (CCCP) enhanced bacterial survival, indicating that A. hydrophila triggered mitophagy to maintain its intracellular survival. Collectively, A. hydrophila induced PINK1/Parkin-mediated mitophagy via its secreted effectors, thereby mitigating antimicrobial pressure and facilitating its own survival. This provided a novel perspective for understanding the intracellular survival mechanisms of extracellular pathogens.