Chemical Biology & Exposure Sciences, Pacific Northwest National Laboratory, Richland, WA, 99352, USA.
The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99163, USA.
Sci Rep. 2019 Feb 4;9(1):1359. doi: 10.1038/s41598-018-37656-7.
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.
哺乳动物肠道中的微生物群在控制宿主生理学方面发挥着动态作用。肠道微生物群活性对宿主健康的影响在胆汁稳态中尤为明显。胆汁由宿主产生，并被肠道微生物群修饰，这影响了总胆汁酸池的净疏水性，并调节宿主信号通路。微生物群修饰胆汁的一个关键机制是通过胆汁盐水解酶（BSH）的酶促活性使胆汁盐去共轭，这被认为是所有进一步微生物代谢的前提。肠道中的 BSH 活性在很大程度上被认为对宿主有益，并且在许多非处方益生菌中发现的许多分类群的基因组中都发现了编码 BSH 的基因。尽管这种酶具有治疗相关性，但目前还没有用于连续监测 BSH 活性的敏感且简单的测定方法，也没有非破坏性的方法来表征其在全细胞或微生物群落样本中的活性。在此，我们描述了一种连续荧光测定法，可用于用纯化蛋白、细胞裂解物、全细胞以及人类肠道微生物组样本对 BSH 活性进行特征描述。该方法是一种“开启”报告策略，它使用合成底物，在 BSH 依赖性转化后产生荧光产物。该测定法用于首次对 BSH 活性进行体内表征。我们还证明了在含有重组 BSH 的人类粪便微生物组样本中连续、非破坏性地定量 BSH 活性。
