Vibrio vulnificus MARTX cytotoxin triggers necroptosis-driven macrophage cell death

Vibrio vulnificus , a gram-negative bacterium, commonly infects humans via contaminated seafood or wounds and can cause severe sepsis. The multifunctional autoprocessing repeat-in-toxin (MARTX) is one of the most crucial virulence factors in V. vulnificus , associated with host pathogenesis. V. vulnificus MARTX induces serious cell damage, leading to subsequent cell death. However, the specific modalities of cell death are diverse and not fully understood. Murine macrophage cells were challenged with V . vulnificus wild-type and Δ rtxA1 strains. Necroptotic events were assessed through western blotting, cytokine production assays, and fluorescence-based analyses. In this study, we showed that MARTX is essential for V. vulnificus -induced macrophage necroptosis as it activates receptor-interacting protein kinase 3 (RIPK3)/mixed-lineage kinase domain-like pseudokinase (MLKL) signaling. In contrast, the MARTX-deficient mutant (ΔrtxA1 ) substantially enhanced phagocytosis by macrophages and abolished necroptosis. We further found that MARTX elicited MLKL mobilization to membrane lipid rafts in response to necroptosis. Disruption of lipid rafts using cholesterol depletion/usurping agents significantly alleviated V. vulnificus -induced cell death. Our findings reveal that V . vulnificus MARTX induces macrophage cell death via the necroptosis pathway, dependent on adequate cholesterol levels and potentially involving other cell death mechanisms.