β-Catenin Elicits Drp1-Mediated Mitochondrial Fission Activating the Pro-Apoptotic Caspase-1/IL-1β Signalosome in Aeromonas hydrophila-Infected Zebrafish Macrophages

Canonical Wnt signaling plays a major role in regulating microbial pathogenesis. However, to date, its involvement in A. hydrophila infection is not well known. Using zebrafish (Danio rerio) kidney macrophages (ZKM), we report that A. hydrophila infection upregulates wnt2, wnt3a, fzd5, lrp6, and β-catenin (ctnnb1) expression, coinciding with the decreased expression of gsk3b and axin. Additionally, increased nuclear β-catenin protein accumulation was observed in infected ZKM, thereby suggesting the activation of canonical Wnt signaling in A. hydrophila infection. Our studies with the β-catenin specific inhibitor JW67 demonstrated β-catenin to be pro-apoptotic, which initiates the apoptosis of A. hydrophila-infected ZKM. β-catenin induces NADPH oxidase (NOX)-mediated ROS production, which orchestrates sustained mitochondrial ROS (mtROS) generation in the infected ZKM. Elevated mtROS favors the dissipation of the mitochondrial membrane potential (ΔΨ(m)) and downstream Drp1-mediated mitochondrial fission, leading to cytochrome c release. We also report that β-catenin-induced mitochondrial fission is an upstream regulator of the caspase-1/IL-1β signalosome, which triggers the caspase-3 mediated apoptosis of the ZKM as well as A. hydrophila clearance. This is the first study suggesting a host-centric role of canonical Wnt signaling pathway in A. hydrophila pathogenesis wherein β-catenin plays a primal role in activating the mitochondrial fission machinery, which actively promotes ZKM apoptosis and helps in containing the bacteria.