Abstracts

Bile salt hydrolases (BSH) are a family of microbial enzymes that play important roles in a wide range of host metabolic processes. They catalyze the hydrolysis of amide bonds in conjugated primary bile acids (BAs), resulting in the release of free amino acids (glycine or taurine) and deconjugated primary BAs. Once deconjugated, free primary BAs can be metabolized to secondary BAs, involved in different intestinal pathologies such as colorectal cancer (CRC). To uncover BSH abundance and its activity in the gut microbiota, a few methods such as biochemical assays and metagenomic analysis are currently used; however, these approaches have several limitations. In this context, we developed a rapid and cost-effective bioluminescence (BL) bioassay for the screening of BSH activity in human feces using an amino luciferin” caged” with the primary BA chenodeoxycholic acid (aLuc-CDCA), thus mimicking the conjugated CDCA with glycine [1]. Since aLuc- CDCA isn’t a substrate for the luciferase enzyme being “caged” by the amidation, the reaction occurs after the BSH-catalyzed hydrolysis that allows the release of the “free” amino luciferin that in turn induces the BL reaction in the presence of luciferase and ATP. Firstly, we investigated in vitro the deconjugation level of aLuc-CDCA (50 µM) in the presence of pure BSH (range 0-10 µg/mL) using different exposure times (15, 30, and 60 min) at 37°C. The BL reaction was triggered, adding in the solution the firefly luciferase (0.5 mg/mL) in Tris-HCl 0.1 mM, pH 7.5, supplemented with Mg₂SO₄ (80 mM) and ATP (40 mM). Kinetic profiles showed a linear correlation between BL emission intensity and increased amount of BSH, with an R² of 0.92 after 60 min of incubation (LOD:0.8 µg/mL; LOQ: 1.6 µg/mL ). Next, we moved to real human feces, but the light output decreased markedly probably due to interferents in the fecal material that affect the luciferase activity. To address this issue, we exploited the possibility of using a whole cell-based BL assay in which human colorectal adenocarcinoma cells (Caco2) genetically encoded with a firefly luciferase (Amydetes vivianii), were exposed to solutions containing aLuc-CDCA (50 µM) previously incubated with pure BSH (range 0-100 µg/mL) for 60 min at 37°C. This approach enables the protection of the luciferase enzyme by cell membranes, obtaining a good correlation between BL signal intensity and BSH amount with a LOD of 2.6 µg/mL. As a proof of concept, the whole cell-based assay has been successfully employed for quantifying BSH activity in intact fecal stools (1mg/1ml) obtained from healthy volunteers and patients with adenoma and CRC diagnosis, thus demonstrating the possibility of using BSH as a potential biomarker for CRC prevention and treatment.1. Roda, A. et al. Chemosensors 2021, 9(6), 122.