Bile salt hydrolase profiling by fluorogenic probes in the human gut microbiome

Bile is a digestive fluid produced in the liver and stored in the gallbladder. It participates in absorption of fatty nutrients and vitamins, and aids in elimination of metabolic waste and toxins. The major chemical components of bile are bile salts that, apart from their function in digestion, are also known to participate in cell signaling by binding host farnesoid X (FXR), vitamin D (VDR), and G-protein coupled bile acid (TGR5) receptors. Microbial bile salt hydrolases (BSHs) catalyze bile salt deconjugation, a gatekeeper reaction that is a prerequisite for all subsequent microbial transformations of bile acids. As a result, BSH determines the composition of the bile salt and acid pools, which in turn affects its nutrient absorption and signaling capabilities. BSH profiling remains a challenge due to a paucity of tools that enable scientists to study its function. In this chapter, we discuss current BSH profiling approaches and demonstrate a novel fluorogenic probe-based assay that circumvents laborious and resource intensive BSH quantification methods. Alongside our assay protocol, we provide the reader with a detailed method for microbial cell extraction from fecal matter. We also cover probe validation protocols that can be adapted for Michaelis-Menten analysis with any BSH expressing strain.