Author response: A selective gut bacterial bile salt hydrolase alters host metabolism (2018)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The human gut microbiota impacts host metabolism and has been implicated in the pathophysiology of obesity and metabolic syndromes. However, defining the roles of specific microbial activities and metabolites on host phenotypes has proven challenging due to the complexity of the microbiome-host ecosystem. Here, we identify strains from the abundant gut bacterial phylum Bacteroidetes that display selective bile salt hydrolase (BSH) activity. Using isogenic strains of wild-type and BSH-deleted Bacteroides thetaiotaomicron, we selectively modulated the levels of the bile acid tauro-β-muricholic acid in monocolonized gnotobiotic mice. B. thetaiotaomicron BSH mutant-colonized mice displayed altered metabolism, including reduced weight gain and respiratory exchange ratios, as well as transcriptional changes in metabolic, circadian rhythm, and immune pathways in the gut and liver. Our results demonstrate that metabolites generated by a single microbial gene and enzymatic activity can profoundly alter host metabolism and gene expression at local and organism-level scales. https://doi.org/10.7554/eLife.37182.001 eLife digest The microbiome, the collection of bacteria that live in and on human bodies, has a strong influence on how well the body works. However, the diversity of the microbiome makes it difficult to untangle exactly how it has these effects. For example, it is poorly understood how the hundreds of species of bacteria that live in the gut affect metabolism – the chemical processes that make life possible. But they are known to influence how metabolic diseases like diabetes and obesity develop. When we eat a meal, the body releases compounds called bile acids to help to digest the food. Once the bile acids reach the colon, the bacteria residing there use enzymes to chemically modify the compounds. Imbalances in the resulting pool of over 50 different bile acids may accelerate how quickly people develop metabolic disorders. It is not clear, however, which bile acids have helpful or harmful effects on metabolism. Yao et al. first identified a selective version of a prevalent gut bacterial enzyme called a bile salt hydrolase. This enzyme was then deleted from a common gut bacterium using genetic tools. Finally, Yao et al. colonized mice lacking any bacteria (i.e., germ-free mice) with either the original bacterium or the hydrolase-deleted bacterium. Mice colonized with the hydrolase-deleted bacteria gained less weight on a high fat diet and had lower levels of fat in their blood and liver. These mice also shifted to burning fats instead of carbohydrates for energy. The changes in the bile acid pool produced in mice colonized with hydrolase-deleted bacteria did not only affect metabolism. Yao et al. found differences in the activity of genes important for other biological processes as well, such as those that control circadian rhythms and immune responses. Further research is needed to investigate whether limiting the activity of the bile salt hydrolase enzyme has similar effects in humans. If so, developing drugs or probiotics that target the enzyme could lead to new treatments for people with metabolic diseases like obesity and fatty liver disease. Investigating the biological effects of other bacterially modified bile acids may identify other possible treatments as well. https://doi.org/10.7554/eLife.37182.002 Introduction The human gut microbiome is known to play a crucial role in human energy harvest and homeostasis (BäckhedBackhed et al., 2004; Turnbaugh et al., 2006). Lean and obese people harbor different gut bacterial communities, suggesting that developing gut bacterial imbalances may contribute to obesity (Ley et al., 2006; Turnbaugh et al., 2006; Turnbaugh et al., 2008). Importantly, transplantation of the fecal microbiota from obese humans to germ-free (GF) mice has been shown to result in the development of obesity-associated metabolic phenotypes in recipient mice (Ridaura et al., 2013). These studies establish a causal relationship between gut bacteria and host metabolic status. The molecular mechanisms by which gut microbes regulate host metabolism, however, remain largely unknown. This lack of mechanistic understanding regarding the functions of microbial species and their metabolic capabilities has limited the effectiveness of both dietary and therapeutic approaches to improving host physiology (Jia et al., 2008; Wallace et al., 2010). The investigation of microbial metabolite production represents both an important opportunity and a challenge in the search to uncover the causal underpinnings of the effects of gut bacteria on host metabolism. One of the most concrete effects that human-associated bacteria have on the host is the production of small molecule metabolites, some of which accumulate to levels in the body higher than that of a typical drug (Donia and Fischbach, 2015). Recent research suggests that bacterial metabolites play important roles in host metabolism by regulating host glucose and energy homeostasis (De Vadder et al., 2014; Gao et al., 2009; Todesco et al., 1991). The complexity of gut microbial ecosystems and associated microbial and host-derived microbial metabolites, however, presents significant obstacles on the path to defining how individual compounds elicit specific in vivo effects. Means to control specific metabolites is critical to understanding how these molecules affect host physiology. In this work, we selectively modulate the in vivo levels of bile acids and demonstrate that this controlled alteration of the metabolite pool exerts distinct effects on host physiology. Bile acids are steroidal natural products that are synthesized from cholesterol in the liver and constitute an important part of the molecular environment of a healthy human gut (Ridlon et al., 2006). Upon ingestion of a meal, bile acids are secreted from the liver and gallbladder into the duodenum where, with the activities of pancreatic enzymes, they form micelles that solubilize lipids and fat-soluble vitamins that are otherwise poorly absorbed. Remaining free bile acids are efficiently reabsorbed from the ileum via the action of bile acid transporters and recirculated back to the liver. Approximately 3–5% of bile acids escape enterohepatic recirculation and enter the colon at a rate of 400–800 mg/day, forming a concentrated pool of metabolites (200 to 1000 μM) (Hamilton et al., 2007). In the colon, these molecules are modified by the resident bacteria in near-quantitative fashion, forming a class of on the order of 50 different metabolites called secondary bile acids (Figure 1A). In addition to their role in digestion, many primary and secondary bile acids act as ligands for host nuclear receptors, including the farnesoid X receptor (FXR), the pregnane X receptor (PXR), the vitamin D receptor (VDR), the liver X receptor (LXR) and the G-protein-coupled receptor TGR5 (Fiorucci and Distrutti, 2015; Katsuma et al., 2005; Makishima et al., 2002; Song et al., 2000; Staudinger et al., 2001). By acting as agonists or antagonists for these receptors, bile acids further impact the regulation of glucose tolerance and homeostasis, insulin sensitivity, lipid metabolism, triglyceride and cholesterol levels, and energy expenditure by the host (Fiorucci and Distrutti, 2015; Modica et al., 2010). Additionally, bile acids regulate their own biosynthesis via an FXR-mediated negative feedback mechanism, which affects downstream nutrient availability for the host (Modica et al., 2010). As a result of these interactions, bile acid imbalance has been implicated as having a causal effect in the development of diet-induced obesity (Fiorucci and Distrutti, 2015). Conversely, modification of the bile acid pool by commensal bacteria has been suggested to induce beneficial changes in host metabolism (Joyce et al., 2014). Figure 1 Download asset Open asset Enzymatic activity of gut bacterial bile salt hydrolase (BSH) enzymes. (A) BSH cleave the amide bond linking primary bile acids to taurine or glycine. (B) Structures of the two most abundant murine-conjugated bile acids, tauro-β-muricholic acid (TβMCA) and tauro-cholic acid (TCA). https://doi.org/10.7554/eLife.37182.003 The mechanisms underlying these effects, however, remain largely undefined. Due to the large number of compounds and receptors involved as well as the additional role of bile acids as biological detergents, the in vivo roles of specific bile acids have been difficult to untangle. Our novel approach to deconvoluting the physiological role of structurally distinct bile acids is to control the in vivo activity of selective bacterial bile salt hydrolases (BSH). BSH hydrolyze conjugated bile acids that have been linked to either taurine or glycine by host liver enzymes, revealing unconjugated bile acids (Figure 1A). This deconjugation step occurs prior to subsequent bacterial conversion of primary bile acids (e.g. cholic acid and chenodeoxycholic acid) to secondary bile acids (e.g. deoxycholic acid and lithocholic acid) (Ridlon et al., 2006). Prior work suggests that BSH play a critical role in regulating host metabolism. However, these studies have not yet uncovered how specific bile acid metabolites exert their in vivo effects on host metabolism, and conflicting results have been reported regarding whether BSH activity should be increased or decreased to achieve host metabolic benefits (Joyce et al., 2014; Li et al., 2013). Research efforts to date have either examined correlative relationships between BSH activities, bile acid levels, and metabolic indications (Li et al., 2013) or investigated the metabolic effects of 'unconjugated' versus 'conjugated' groups of bile acids (Joyce et al., 2014). It is imperative to be able to differentiate bile acids in vivo based on their structure in order to understand their effects on host metabolism. As an important example, taurocholic acid (TCA) and tauro-β-muricholic acid (TβMCA) are both conjugated bile acids but exert different physiological effects: TCA is an FXR agonist, while TβMCA is an FXR antagonist (Figure 1B) (Li et al., 2013; Sayin et al., 2013). Herein, we uncover a group of bacteria within the abundant human gut commensal genus Bacteroides that possess selective BSH activity. We then identify the gene responsible for this activity in Bacteroides thetaiotaomicron and construct a knockout strain. By monocolonizing germ-free (GF) mice with the wild-type or BSH-deleted strain, we demonstrate that we can predictably alter the in vivo bile acid pool using this selective enzyme and that this change has significant effects on host metabolic status. Our results demonstrate that the deletion of a single bacterial gene can exert significant effects on host metabolism in a gnotobiotic environment and highlight the importance of modulating specific compounds when seeking to understand the effects of bacterial metabolites on host physiology. Results Selected species of Bacteroides accept distinct bile acid cores as BSH substrates BSH (EC 3.5.1.24) are found across a wide range of bacterial genera from the two dominant gut phyla, Bacteroidetes and Firmicutes (Jones et al., 2008). However, the structural and activity characterization of these enzymes has been largely limited to Gram-positive species (i.e. Clostridia, Lactobacillus, Bifidobacterium, Listeria) (Begley et al., 2006; Rossocha et al., 2005). These enzymes largely demonstrate non-selective activities, cleaving all conjugated bile acids independent of either the bile acid core or amino acid conjugate (taurine or glycine) (Ridlon et al., 2006). While differential reactivity toward conjugated substrates has been observed in some Gram-positive strains, in these cases, the selectivity has been based on a preference for one amino acid over the other, not on the structure of the steroidal core (De Boever P and Verstraete, 1999; Grill et al., 1995; Kim et al., 2004; Ridlon et al., 2006). In contrast, the activity of Gram-negative bacteria has been largely underexplored. While Bacteroides fragilis ATCC 25285 was reported to exhibit non-selective BSH activity (Stellwag and Hylemon, 1976), some Bacteroides vulgatus strains were observed to cleave taurochenodeoxycholic acid (TCDCA) and TβMCA but minimally cleaved TCA (Chikai et al., 1987; Kawamoto et al., 1989), thus exhibiting a degree of selectivity based on the hydroxylation pattern of the steroid. These results suggested to us that perhaps other strains within the phylum Bacteroidetes might display steroidal core-based selectivity. To investigate this question, we performed a screen of the BSH activity of twenty Bacteroidetes strains found in the human gut (Figure 2A and Figure 2—figure supplement 1 and 2) (Kraal et al., 2014). We also tested Clostridium perfringens and Lactobacillus plantarum, two Gram-positive species with known non-selective BSH activities, for comparison. We incubated pre-log phase cultures of individual strains with a group of either the most abundant tauro- or the most abundant glyco-conjugated bile acids found in the human and murine GI tracts. We monitored deconjugation over time by UPLC-MS and determined that all hydrolysis reactions had reached steady state by 48 hr (Figure 2B, Figure 2—figure supplement 3). We then quenched the cultures and profiled bacterial bile acid metabolism. As expected, C. perfringens ATCC 13124, L. plantarum WCFS1, and B. fragilis ATCC 25285 deconjugated all conjugated bile acid substrates tested. Strikingly, the majority of Bacteroidetes strains tested displayed some degree of selectivity for conjugated bile acid substrates, with a preference for deconjugating tauro- over glyco-conjugated substrates. A subset of these strains (B. thetaiotaomicron VPI-5482, B. caccae ATCC 43185, B. fragilis 638R, Bacteroides sp. D2, and Bacteroides sp. 2_1_16; Group I – red, Figure 2A) exhibited selectivity exclusively based on the steroidal core structure, deconjugating C12 = H primary bile acids (i.e. TCDCA, GCDCA, and TβMCA) but not C12 = OH primary bile acids (i.e. TCA and GCA). Figure 2 with 3 supplements see all Download asset Open asset Identification of selective BSH activity in the human gut bacterial phylum Bacteroidetes. (A) Deconjugation ability of twenty prevalent Bacteroidetes strains and two Firmicutes strains found in the human gut represented as heat maps. Individual strains were incubated for 48 hr total with a group of glyco- or tauro-conjugated bile acids found in human and murine GI tracts. G (glyco-), T (tauro-), CA (cholic acid), CDCA (chenodeoxycholic acid), UDCA (ursodeoxycholic acid), DCA (deoxycholic acid), LCA (lithocholic acid), βMCA (β-muricholic acid). Assays were performed in biological duplicate. Group I (red): Bacteroidetes species that deconjugate primary bile acids based on steroidal core structure (C12 = H but not C12 = OH); Group II (gray): species that deconjugate based on amino acid conjugate; Group III (blue): species that deconjugate all bile acid substrates; Group IV (black): no deconjugation observed. (B) Representative UPLC-MS timecourses for deconjugation of TDCA and TLCA showing that steady state has been reached by 48 hr. (C) Representative UPLC-MS traces showing that Bacteroides thetaiotaomicron wild-type (Bt WT) and BtΔ1259 deconjugate TUDCA, whereas BtΔ2086 does not. BtΔ2086,2086 + recovered the deconjugation function while the BtΔ2086,CTRL +control strain containing an empty pNBU2 vector did not, demonstrating that BT2086 is responsible for bile salt hydrolase activity in Bt. (D) Representative UPLC-MS traces showing that Bt WT deconjugates the murine primary bile acid TβMCA but not TCA, whereas BTΔ2086 (Bt KO) does not deconjugate either bile acid. https://doi.org/10.7554/eLife.37182.004 To our knowledge, this study represents the first systematic evaluation of BSH activity in the common gut-bacterial phylum Bacteroidetes. Given that specific conjugated and unconjugated bile acids bind to different host receptors and have the potential to exert different downstream effects, the selectivity uncovered here may have important physiological consequences depending on which Bacteroides species colonize the host. To further explore this possibility and define the effects of selective BSH on host physiology, we monocolonized GF mice with isogenic strains of wild-type and BSH-deleted B. thetaiotaomicron as described below. BT2086 is responsible for BSH activity in B. thetaiotaomicron We recognized that deletion of the BSH enzyme from one of the Group I Bacteroides species would provide us with a paired set of isogenic strains (wild-type and knockout) that would allow us to rationally manipulate the in vivo bile acid pool in a highly specific manner. In mice, the two most abundant primary bile acids are TCA and TβMCA (Sayin et al., 2013). Based on the observed selectivity for deconjugating C12 = H but not C12 = OH core primary bile acids, we predicted that colonization with a BSH wild-type strain would result in lower levels of TβMCA (C12 = H) relative to knockout colonized mice, while the levels of TCA (C12 = OH) in both groups would remain constant. All the five Group I strains displayed weak-to-moderate deconjugation of TβMCA in vitro (Figure 2A). Importantly, we did not detect any products of TCA deconjugation from any of these strains. This result suggested that the levels of deconjugated CA in mice colonized with these bacteria would remain low to undetectable, while the levels of deconjugated βMCA could build up due to enterohepatic recirculation. We decided to focus our efforts on generating paired isogenic strains in one of these species, B. thetaiotaomicron (Bt). Although this strain displayed relatively weak TβMCA-deconjugating activity, Bt had been previously shown to be amenable to genetic manipulation, allowing knockout of putative BSH genes (Cullen et al., 2015; Koropatkin et al., 2008). We performed a BLASTP search of the characterized BSH from C. perfringens (Ridlon et al., 2006) against the Bt genome and identified two genes, BT2086 and BT1259, as putative BSH. We constructed unmarked deletions of these genes using allelic exchange and then tested the resultant mutants for their ability to deconjugate bile acids in whole cell culture using UPLC-MS. The BtΔ2086 mutant (henceforth referred to as Bt KO) had lost the ability to cleave conjugated bile acid substrates. In contrast, the BtΔ1259 mutant displayed no loss-of-function phenotype (Figure 2C). Complementation of the Bt KO strain with BT2086 restored BSH activity (Figure 2C), confirming that BT2086 is necessary for bile acid deconjugation in Bt. Since bile salt hydrolases and penicillin V amidases (PVA) both belong to the cholylglycine hydrolase (CGH) family and share a high degree of sequence homology, it is possible that BT1259 is a PVA, although additional experiments would be needed to definitively establish this activity (Jones et al., 2008; Panigrahi et al., 2014). Finally, we verified that when incubated with both TβMCA and TCA, Bt wild-type (Bt WT) deconjugated TβMCA but not TCA, whereas the Bt KO strain did not deconjugate either bile acid (Figure 2D). Bacteroidetes BSH exhibit evolutionary diversity A phylogenetic grouping of the 20 Bacteroidetes strains assayed revealed that while the species that deconjugate bile acids based on the amino acid conjugate (Group II – gray, Figure 2A) form a partial clade (Figure 3A), the strains that exhibit selectivity based on the steroid core (Group I – red) and those that display no selectivity (Group III – blue) are not separated into distinct clades. A BLAST-P search using BT2086 as a query gene identified candidate BSH genes in 19 of the 20 Bacteroidetes strains tested. Bacteroides finegoldii DSM 17565 did not display BSH activity and also lacked a putative BSH. A phylogenetic tree resulting from the multiple sequence alignment of these 19 candidate BSH genes revealed a lack of homology among enzymes within a given activity group (Figure 3B). Group II enzymes, which had formed a clade at the strain level, are now separated into two groups, and steroid core-selective strains (Group I) do not cluster significantly. Taken together, these that preference for C12 = H over C12 = OH primary bile acid cores is an activity that may have multiple from of the BSH Figure 3 Download asset Open asset of Bacteroidetes strains and putative BSH (A) tree of 20 Bacteroidetes strains using whole Bacteroidetes strains from Group II deconjugation based on amino acid) form a partial while Group I and Group III strains do not into distinct clades. (B) tree of candidate Bacteroidetes BSH A search for BLAST-P of BT2086 identified an in 19 of the 20 Bacteroidetes species to the the of in which this was reached in a of 1000 significant of Bacteroidetes strains into based on enzymatic activity was observed. number of of Bt BSH results in specific changes to murine bile acid in vivo To our that a single bacterial the bile salt hydrolase would result in a and selective alteration of the in vivo bile acid GF mice were monocolonized with Bt WT or Bt KO Figure To further effects of this single microbial gene on host metabolism and energy we also performed an in using groups of mice with Bt mice with Bt KO or GF control mice which Figure For both over a mice were a diet to a human diet For the of the mice were from gnotobiotic to metabolic with in the in order to metabolic status. Figure with 2 supplements see all Download asset Open asset of GF mice with WT and Bt KO results in predictably altered bile acid (A) germ-free mice were monocolonized with either Bt WT or KO and a diet for In a monocolonized mice (Bt WT or Bt KO) or GF mice were a for and to the to metabolic and (B) Bile acid using UPLC-MS revealed that Bt mice displayed higher levels of TβMCA in than Bt mice while the levels of TCA the between the two βMCA levels were higher in Bt and no CA was in any group changes were observed in (C) and ileum although no βMCA was observed in any group in the Data are as acid). For the = mice For the = mice by multiple We first that BT2086 was in vivo by on from Bt mice (Figure supplement As expected, no BT2086 were in the of mice. We then performed bile acid on and blood from mice in both As we Bt mice displayed higher levels of TβMCA in than Bt mice in the (Figure Bt mice also exhibited lower levels of βMCA the of TβMCA than Bt mice. Importantly, the levels of TCA between the two groups, and no CA was in either These results are with our in vitro data showing that the Bt BSH can deconjugate C12 = H but not C12 = OH primary bile We observed the significant in βMCA levels in (Figure highlight In the in with (Sayin et al., GF mice had higher bile acid levels than colonized mice Bt WT KO with the of Bt mice displayed lower levels of βMCA Bt WT GF and KO than of Bt mice, while CA in both groups (Figure We also profiled the bile acid in the the of bile acid from the small in the As expected, the bile acid were higher in this than in and fecal (Figure (Sayin et al., 2013). We observed the as we had in with higher TβMCA levels in Bt mice, although the differences were not significant we that the of this was not the of the small This of in the ileum may help the large range of bile acid observed in this In to the and the liver and (Figure supplement 2) of Bt and Bt mice similar bile acid with no significant differences These data are with that the differences between GF and mice were in the and colon, not in the liver or the blood (Sayin et al., 2013). We also observed a significant of bile acid genes in the liver suggesting that bile acid may the observed differences between the two Taken together, our data that we can rationally manipulate the in vivo bile acid pool in the and to a in the small and colon (i.e. using a Bacteroides BSH enzyme that selectively C12 = H but not C12 = OH conjugated primary bile Importantly, this selective hydrolysis us to modulate the levels of a known FXR while the levels of TCA, an FXR Bt BSH affects host metabolic indications shown that Bt BSH selectively of the bile acid pool in monocolonized GF mice, we to explore how these specific changes in bile acid levels host metabolism. Strikingly, Bt mice gained less weight on the diet than Bt mice in the (Figure This result is it has been shown that GF mice are to weight gain when a diet et al., 2007). In we performed a relatively diet to other studies that have to study metabolic changes et al., 2015; et al., 2014; et al., et al., and we did not to significant changes in body weight over the of a Importantly, the host effects observed are not due to differences in colonization In both Bt WT and Bt KO efficiently colonized the GI and the only bacterial species in the (Figure and Figure supplement These data that the observed metabolic changes are due to in the bile acid pool by the or of the Bt BSH. Figure Download asset Open asset Bt BSH colonization affects weight gain and lipid (A) germ-free mice monocolonized with Bt KO gained less weight a diet challenge than Bt mice. = mice multiple using the = (B) did not between the Bt WT and Bt KO strains in GF mice. For the 2 were to For the 2) were at were also to of monocolonized the were in the GF = levels in and (C) free fatty and