RESUMEN
Diurnal rhythmicity of cellular function is key to survival for most organisms on Earth. Many circadian functions are driven by the brain, but regulation of a separate set of peripheral rhythms remains poorly understood. The gut microbiome is a potential candidate for regulation of host peripheral rhythms, and this study sought to specifically examine the process of microbial bile salt biotransformation. To enable this work, an assay for bile salt hydrolase (BSH) that could work with small quantities of stool samples was necessary. Using a turn-on fluorescence probe, we developed a rapid and inexpensive assay to detect BSH enzyme activity with concentrations as low as 6-25 µM, which is considerably more robust than prior approaches. We successfully applied this rhodamine-based assay to detect BSH activity in a wide range of biological samples such as recombinant protein, whole cells, fecal samples, and gut lumen content from mice. We were able to detect significant BSH activity in small amounts of mouse fecal/gut content (20-50 mg) within 2 h, which illustrates its potential for use in various biological/clinical applications. Using this assay, we investigated the diurnal fluctuations of BSH activity in the large intestine of mice. By using time restricted feeding conditions, we provided direct evidence of 24 h rhythmicity in microbiome BSH activity levels and showed that this rhythmicity is influenced by feeding patterns. Our novel function-centric approach has potential to aid in the discovery of therapeutic, diet, or lifestyle interventions for correction of circadian perturbations linked to bile metabolism.
Asunto(s)
Amidohidrolasas , Ácidos y Sales Biliares , Animales , Ratones , Fluorescencia , Amidohidrolasas/metabolismo , Ritmo CircadianoRESUMEN
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.
Asunto(s)
Microbioma Gastrointestinal , Amidohidrolasas/metabolismo , Ácidos y Sales Biliares , Heces , HumanosRESUMEN
Animals produce bile to act as an antibacterial agent and to maximize the absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also participate in signaling pathways by modulating physiological processes upon binding host receptors. Upon excretion of bile salts from the gall bladder into the intestine, the gut microbiota can create metabolites with modified signaling capabilities. The category and magnitude of bile salt metabolism can have positive or negative effects on the host. A key modification is bile salt hydrolysis, which is a prerequisite for all additional microbial transformations. We have synthesized five different fluorogenic bile salts for simple and continuous reporting of hydrolysis in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced primary bile salts, but some microbially modified secondary bile salts also display significant turnover.