A continuous fluorescence assay for simple quantification of bile salt hydrolase activity in the gut microbiome

Jacqueline M Weaver,Aaron T Wright,Kristoffer R Brandvold,Christopher Whidbey

doi:10.1038/s41598-018-37656-7

Jacqueline M Weaver, Aaron T Wright + Show 2 more

Open Access

https://doi.org/10.1038/s41598-018-37656-7

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Abstract

The microbiota of the mammalian gut plays a dynamic role in controlling host physiology. The effect of gut microbiota activity on host health is particularly evident in the case of bile homeostasis. Bile is produced by the host and is modified by the gut microbiota, which impacts the net hydrophobicity of the total bile acid pool, and also modulates host signaling pathways. A key mechanism by which the microbiota modify bile is through deconjugation of bile salts through bile salt hydrolase (BSH) enzymatic activity, which is postulated to be a prerequisite for all further microbial metabolism. BSH activity in the gut is largely considered to be beneficial for the host, and genes encoding BSHs are found in the genomes of many taxa found in over-the-counter probiotics. Despite the therapeutic relevance of this enzyme, there is no sensitive and simple assay for continuous monitoring of BSH activity, and there are no non-destructive means of characterizing its activity in whole cell or microbial community samples. Herein, we describe a continuous fluorescence assay that can be used for characterization of BSH activity with purified protein, cell lysates, whole cells, and in human gut microbiome samples. The method is a “turn-on” reporter strategy, which employs synthetic substrates that yield a fluorescent product upon BSH-dependent turnover. This assay is used to show the first in vivo characterization of BSH activity. We also demonstrate continuous, non-destructive quantification of BSH activity in a human fecal microbiome sample containing recombinant BSH.

Highlights

The animal gut accommodates a complex mixture of microbes that greatly influence host health, with effects including alteration of nutrient absorption, creation of chemical signals that modulate host physiology, and protection against pathogens[1,2]
Deconjugation of bile salts results from the activity of microbial bile salt hydrolases (BSHs), which hydrolyze the amide bond between the bile acid and glycine/taurine (Fig. 1A) using a nucleophilic cysteine[3,22]
Increasing BSH activity has been suggested to be an effective strategy for treating obesity and hypercholesterolemia, because if bile acids are excreted in excess, endogenously produced cholesterol pools will be depleted to compensate for the production of fresh bile[13]

Summary

Introduction

The animal gut accommodates a complex mixture of microbes that greatly influence host health, with effects including alteration of nutrient absorption, creation of chemical signals that modulate host physiology, and protection against pathogens[1,2]. Fluorescent bile acid analogs have been previously reported[33,34,35,36], and were used for characterization of bile acid movement in the gut or other applications, but have not been applied to a kinetic activity-based assay for BSH. Spectrographic characterization of the product revealed that, similar to the analogous protease substrates, the conjugated probe was much less fluorescent relative to the free aminocoumarin when examined at identical excitation and emission wavelengths (Fig. 2A), and had potential to act as a reporter of BSH activity.

Results

Conclusion