Bile salt hydrolase catalyses formation of amine-conjugated bile acids

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1–6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

data that indicates that 100 uM BBAAs is a physiologically relevant concentration?Minor Point: The point of the RNA-Seq is unclear, since there doesn't seem to have been an attempt to look for potential candidate conjugating enzymes apart from BSH.It is well known that bsh is typically constrictively expressed in most bacterial species and strains.In the enteroid model, the authors note a discrepancy in receptor agonism with the luciferase assay.It is known that human carboxypeptidases from pancreas cleave non-canonical amino acid-bile acid conjugates (PMID: 2867000).Enteroendocrine cells of the ileum express carboxypeptidases (PMID: 25051500).Did the authors check to see if the BBAAs added to enteroids remained intact at the end of the experiment?D. Appropriate use of statistics and treatment of uncertainties E. Conclusions: robustness, validity, reliability Unless the authors can physiologically justify the use of BBAAs at 100 uM, the conclusion that "BBAAs may unexpectedly impact the metabolism of substrates, drugs, and carcinogens regulated by PXR and AHR" (line 224-225) is a stretch.Similar point at line 240 that "BBAAs facilitate communication between the microbiota and host…" G. Clarity and context: lucidity of abstract/summary, appropriateness of abstract, introduction and conclusions The manuscript and figures are clearly presented.
Referee #2 (Remarks to the Author): This manuscript the authors establish bacterial bile salt hydrolase as a bile acid amidating enzyme (an amine N-acyltransferase with bile acid as carboxylate co-substrate).This is an interesting manuscript: the experiments are thorough and the combination of biochemistry and genetic work make a compelling case that this enzyme is responsible for making BBAAs in a variety of bacterial taxa.
The organoid assay is nice and its points of disagreement with the luciferase-based NHR activation assay are a cautionary note on the latter.My only point of concern is that I don't think these experiments go far enough in establishing the physiologic relevance of the BBAAs.Not required, but I would strongly suggest that the authors test, e.g., wild-type vs mutant B. fragilis in a germ-free mouse (mono-colonized or in the context of a small defined community) to show an effect on the host -almost any reasonable/convincing effect would do.
Referee #3 (Remarks to the Author): In this paper, Rimal and colleagues aimed at exploring the metabolism of bile acid by intestinal bacteria.They observed that bile salt hydrolase (BSH) exhibits an amine N-acyl transferase activity that conjugates amines to form bile acid amidates (BBAAs).Using different methods the authors demonstrated that BSH is required for BBAA production and that BBAAs is able to activate several host ligand-activated transcription factors including FXR, PXR, androstane receptor, and AhR.
The paper is clear and well written.I do not see any methodological issue.The in vitro demonstration is convincing but human data and effects in vivo and in pathological models would reinforce the paper.
here also express carboxypeptidase E (data not shown).We appreciate the reviewer's concern that in some cases, BBAAs could be de-amidated by active carboxypeptidase E; however, quantitatively measuring this activity in detail would entail overcoming major technical hurdles working with enteroids including access to small amounts of material, challenges dissociating metabolites from the matrigel substrate, as well as how the culturing conditions may influence metabolism of BBAAs.There are also potential differences between the reporter lines and the enteroids including uptake/transport, metabolism, and receptor levels.Our goal by including this data was to demonstrate that BBAAs could activate host receptors.Since our main focus was on establishing a new enzymatic activity for BSH, we now include the reporter and enteroid data as extended data.Further, we include the following in the manuscript: "However, the impact of other host and/or bacterial enzymes on BBAA metabolism and associated activity warrants further investigation." Referee #2 (Remarks to the Author): This manuscript the authors establish bacterial bile salt hydrolase as a bile acid amidating enzyme (an amine N-acyltransferase with bile acid as carboxylate co-substrate).This is an interesting manuscript: the experiments are thorough and the combination of biochemistry and genetic work make a compelling case that this enzyme is responsible for making BBAAs in a variety of bacterial taxa.
The organoid assay is nice and its points of disagreement with the luciferase-based NHR activation assay are a cautionary note on the latter.My only point of concern is that I don't think these experiments go far enough in establishing the physiologic relevance of the BBAAs.Not required, but I would strongly suggest that the authors test, e.g., wild-type vs mutant B. fragilis in a germ-free mouse (mono-colonized or in the context of a small defined community) to show an effect on the host -almost any reasonable/convincing effect would do.

Response:
We thank the reviewer for this helpful advice.We have addressed these concerns in two ways.First, as suggested, we conducted the germ-free experiment (see Figure 2f-j) where we have colonized germ-free recipients with either wildtype or Δbsh B. fragilis.We noted that in mice colonized with Δbsh B. fragilis there was a significant reduction in total BBAAs as well as significantly reduced levels of primary bile acids and free taurine.Additionally, to help provide more human physiological relevance, we examined the levels of BBAAs in human newborns and tracked these levels through the first year of life.We found that BBAAs are found in newborn stool samples early in life and that these levels are consistent with colonization of the newborn gut with bsh-expressing bacteria.These new data strongly support that BSH is responsible for BBAA production.
Referee #3 (Remarks to the Author): In this paper, Rimal and colleagues aimed at exploring the metabolism of bile acid by intestinal bacteria.They observed that bile salt hydrolase (BSH) exhibits an amine N-acyl transferase activity that conjugates amines to form bile acid amidates (BBAAs).Using different methods the authors demonstrated that BSH is required for BBAA production and that BBAAs is able to activate several host ligand-activated transcription factors including FXR, PXR, androstane receptor, and AhR.
The paper is clear and well written.I do not see any methodological issue.The in vitro demonstration is convincing but human data and effects in vivo and in pathological models would reinforce the paper.

Response:
We thank the reviewer for this helpful advice.Please see our response to reviewer 2 above addressing the addition of human data.