Multiple-sequence alignment of vertebrate Bid. The Bid from each species were aligned using the Clustal O program and decorated with BoxShade software. The conserved BH3 domains were boxed. The caspase-8 cleavage site (LETD), the granzyme B cleavage site (HELQ), and the calpain cleavage site was marked by red arrow, blue arrow, and green arrow, respectively.

Phylogenetic tree analysis of vertebrates Bid. The phylogenetic tree was constructed using Mega X software using N-J method. Bootstrap was calculated with 10,000 repeats. The tree is divided into four main clades (clade A–D), with clade (A) composed of fish Bid, clade (B) composed of amphibian Bid, clade (C) composed of birds Bid and clade (D) composed of mammalian Bid. Accession numbers for sequences were listed following the common names of species. Zebrafish Bid was marked by triangle in the tree.

Expression analysis of zebrafish bid in normal embryos and embryos infected with E. ictaluri by using qPCR. (A) Expression of bid in normal embryos. Thirty to 50 embryos were collected for each time point. Expression of bid was normalized to the housekeeping gene gapdh.(B) Expression of bid in embryos infected with E. ictaluri. The zebrafish larvae at 4 dpf were infected with 2 × 10⁸ CFU/ml E. ictaluri and were collected at 12, 24, and 48 h postinfection (hpi). The fold changes of bid in embryos infected with E. ictaluri at each time point were analyzed using the 2^-ΔΔCT method relative to the corresponding control group. Asterisks indicated that the expression level of bid in embryos infected with E. ictaluri was significantly higher than that of the control. **p < 0.01.

Apoptotic activity of zebrafish Bid at the early apoptosis stage (A) and the later apoptosis stage (B). EPC cells were transiently transfected with a 2 μg p3xFLAG or Bid-FLAG plasmid. At 24 hours post-transfection, the cells were infected with E. ictaluri with an MOI of 1, 5, and 10 at 28°C. After infection for 1 h, the extracellular bacteria were killed using gentamicin (50 µg/mL). Apoptosis at 6 hpi was detected using an Annexin V-FITC/PI Apoptosis Detection Kit on the CytoFLEX LX flow cytometer. *p< 0.05.

Figure 5 Antibacterial activity of zebrafish Bid in vitro (A) and in vivo (B). For in vitro bacterial invasion assays (A), the EPC cells were transiently transfected with 500 ng p3×FLAG or Bid-FLAG plasmid. At 24 h post-transfection, the cells were infected with E. ictaluri at an MOI of 10. At 1.5 h post-infection, the extracellular bacteria were killed using gentamicin (50 µg/ml). At 3 and 6 hpi, the cells were lysed, and the intracellular bacterial colony-forming units (CFU) were calculated. For in vivo bacterial invasion assays (B), the fertilized eggs were microinjected with 200 ng/μl p3×FLAG or Bid-FLAG. At 4 dpf, the larvae were infected with E. ictaluri at an MOI of 10. At 48 hpi, the fish were ground, and the CFU was calculated. *p < 0.05, **p < 0.01.

Expression analysis of apoptosis-related genes in bid-overexpressed embryos without (A–C) or with (D–F)E. ictaluri infection. The fertilized eggs were microinjected with 200 ng/μl p3×FLAG empty plasmid or Bid-FLAG plasmid. The microinjected eggs were raised to 4 dpf, and the expressions of apoptosis-related genes were examined using qPCR. The 4-dpf microinjected larvae were infected with E. ictaluri, and the expressions of apoptosis-related genes were examined using qPCR at 12 hpi. The expression levels of target genes were analyzed using the 2^-ΔΔCT method by respectively calculating with the Ct values of gapdh(A, D), EF-1α(B, E), and the average Ct of gapdh and EF-1α(C, F). The average Ct values of the two reference genes are listed in Table S2. *p < 0.05, **p < 0.01. ns, no significance.

The effects of Bid and p53 on fish survival (A) and the number of E. ictaluri in larvae (B). For fish survival analysis, the fertilized eggs were microinjected with p3×FLAG empty plasmid, p53-Flag, or co-microinjected p53-Flag and bid-Flag and raised to 4 dpf, and then infected with E. ictaluri for 6 days. The numbers of surviving larvae were counted daily, and survival curves were generated. The number of E. ictaluri in larvae was calculated as the in vivo bacterial invasion assays. **p < 0.01.

Antibacterial activity of Bid is caspase-8 independent. (A) Survival rates for larvae treated with Z-IETD-FMK. (B) Number of E. ictaluri in larvae treated with Z-IETD-FMK. (C) Number of E. ictaluri in larvae microinjected with Bid-Flag and treated with Z-IETD-FMK. **p < 0.01.

Mechanism of the antibacterial activity of full-length Bid.