Hemolysin EthA is an Edwardsiella T3SS effector that induces PANoptosis in macrophages.

Edwardsiella piscicida is an enteric intracellular bacterial pathogen. It has been reported that this bacterium produces two types of hemolysins: the secreted, pore-forming toxin HlyA and the cell-associated toxin EthA. EthA has been reported to enter epithelial cells by binding to outer membrane vesicles (OMVs) and that once inside the host cells, it releases lipopolysaccharide from the OMVs to induce pyroptosis. We report here the existence of another pathway by which EthA is secreted and delivered into host cells: the type III secretion system (T3SS) of E. piscicida. The ΔFur box-PethB strain that highly expresses EthA was constructed by deleting the binding motif of Fur in front of the ethB-ethA operon. The ΔFur box-PethB strain induces PANoptosis (a combination of pyroptosis, apoptosis and necroptosis) in a T3SS-dependent manner in murine macrophages. The highly expressed EthA stimulates pyroptosis partially via the NLRP3 inflammasome and concomittantly induces apoptosis and necroptosis, as evidenced by elevated ratios of positive cells in TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, elevated protein levels of cleaved caspase-3 and phosphorylated MLKL. PANoptosis is characterized by cell swelling, membrane rupture and cell blebbing. Additionally, the highly expressed EthA inhibits the phosphorylation of the serine/threonine kinase TAK1 and transcription factor p65, preventing the nuclear translocation of phosphorylated p65 into murine macrophages. Overall, EthA has been identified as a novel T3SS substrate in E. piscicida. Its high expression inhibits the phosphorylation of TAK1, thereby increasing PANoptosis and blocking the inflammatory pathway.