Recombinant Bile Salt Hydrolase Enhances the Inhibition Efficiency of Taurodeoxycholic Acid againstVirulence.

Abstract

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 ofgrowth, hydrogen sulfide (HS) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select thegene for cloning. Thegene fromwas PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein inBL21 and168 (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 fromwas analyzed by a Dot-Blot.-BSH was evaluated for the inhibition ofgrowth.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 reducedHS production and the virulence gene expression of,,, and. BSH activity was observed inandunder anaerobe but notunder 10% COair. After the sequence alignment offrom ten bacteria,fromwas 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 subclonedand amylase signal peptide sequence was inserted into pDR-BSH,was transformed and named-BSH. The transformation was evaluated using PCR witharound 3 kb andBSH 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 againstvirulence, and recombinant secretory BSH from-BSH reducedgrowth, suggesting a new potential intervention against the pathogen-induced chicken NE.