is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen . Assays were conducted to evaluate the inhibition of growth, hydrogen sulfide (HS) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the gene for cloning. The gene from was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR... More
is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen . Assays were conducted to evaluate the inhibition of growth, hydrogen sulfide (HS) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the gene for cloning. The gene from was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in BL21 and 168 (BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from was analyzed by a Dot-Blot. -BSH was evaluated for the inhibition of growth. growth reached 7.8 log10 CFU/mL after 24 h culture. growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced HS production and the virulence gene expression of , , , and . BSH activity was observed in and under anaerobe but not under 10% CO air. After the sequence alignment of from ten bacteria, from was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned and amylase signal peptide sequence was inserted into pDR-BSH, was transformed and named -BSH. The transformation was evaluated using PCR with around 3 kb and BSH around 5 kb. Secretory BSH expressed from -BSH was determined for His-tag using Dot-Blot. Importantly, growth was reduced greater than 59% log10 CFU/mL in the -BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against virulence, and recombinant secretory BSH from -BSH reduced growth, suggesting a new potential intervention against the pathogen-induced chicken NE.
