Identification and functional analysis of Bcl2-associated agonist of cell death (Bad) reveals its pro-apoptotic and antibacterial roles in Cromileptes altivelis

Abstract

Bcl-2-associated agonist of cell death (Bad) is a pro-apoptotic member of the Bcl-2 family that contains only the BH3 domain and plays a critical role in mitochondrial apoptosis in mammals. However, its functional role in teleosts remains to be fully elucidated. In this study, we identified and characterized the Bad homolog from Cromileptes altivelis (designated CaBad). The complete coding sequence of CaBad comprises 504 bp, encoding a protein of 167 amino acids with only one conserved BH3 domain. CaBad was ubiquitously expressed in healthy C. altivelis, with highest transcript levels detected in immune-related tissues including liver, spleen, and head kidney. Following challenge with Vibrio harveyi, CaBad expression was significantly upregulated in the liver, spleen, and head kidney. Subcellular localization analysis revealed that CaBad was distributed in the cytoplasm and nucleus, and showed enhanced mitochondrial translocation during apoptosis. Flow cytometry assays demonstrated that CaBad promoted apoptosis of C. altivelis brain (CAB) cells upon V. harveyi flagellin B (Fla B) stimulation. This pro-apoptotic effect was mediated through the mitochondrial pathway, as demonstrated by loss of mitochondrial membrane potential, increased DNA fragmentation, activation of Caspase 9 and Caspase 3, and upregulation of key pro-apoptotic proteins including cytochrome c and p53. Furthermore, CaBad was found to inhibit the NF-κB signaling pathway, as evidenced by suppressed IKKα phosphorylation and downregulated transcription of NF-κB subunits and pro-inflammatory cytokines both in vitro and in vivo. Importantly, in vivo infection experiments showed that CaBad overexpression significantly reduced bacterial loads in immune tissues, whereas CaBad knockdown led to increased bacterial colonization. Collectively, our results indicate that CaBad exerts a conserved pro-apoptotic function via the mitochondrial pathway and contributes to antibacterial immunity in C. altivelis, providing new insights into the role of BH3-only proteins in teleosts host defense.

Keywords: Antibacterial immunity; Apoptosis; Bad; Cromileptes altivelis; Mitochondria.