Bile salt hydrolases shape the bile acid landscape and restrict Clostridioides difficile growth in the murine gut

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.

The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments of the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain The following table lists non-polymeric compounds, carbohydrate monomers and non-standard Full wwPDB X-ray Structure Validation Report 7SVG residues in protein, DNA, RNA chains that are outliers for geometric or electron-density-fit criteria: Mol Type Chain Res Chirality Geometry Clashes Electron density 2 TAU A 501 -X --2 Entry composition i ○ There are 4 unique types of molecules in this entry. The entry contains 11398 atoms, of which 117 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Choloylglycine hydrolase. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

HIS HIS HIS HIS
• Molecule 1: Choloylglycine hydrolase Chain B:

HIS HIS HIS HIS HIS
• Molecule 1: Choloylglycine hydrolase Chain C:  Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 4.16% of the height of the origin peak. No significant pseudotranslation is detected.
5 Model quality i ○

Standard geometry i ○
Bond lengths and bond angles in the following residue types are not validated in this section: OCS, TAU, JN3 The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond length outliers.

Mol Chain
There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively. The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity. The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry-related clashes. The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 2.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All (45)  There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. There are no Ramachandran outliers to report. 7SVG

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. There are no chirality outliers.

Mol Chain
All (9) torsion outliers are listed below: Mol Chain Res Type Atoms There are no ring outliers.
3 monomers are involved in 7 short contacts: There are no chirality outliers.
All (17)  The following is a two-dimensional graphical depiction of Mogul quality analysis of bond lengths, bond angles, torsion angles, and ring geometry for all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the validation Tables will also be included. For torsion angles, if less then 5% of the Mogul distribution of torsion angles is within 10 degrees of the torsion angle in question, then that torsion angle is considered an outlier. Any bond that is central to one or more torsion angles identified as an outlier by Mogul will be highlighted in the graph. For rings, the root-mean-square deviation (RMSD) between the ring in question and similar rings identified by Mogul is calculated over all ring torsion angles. If the average RMSD is greater than 60 degrees and the minimal RMSD between the ring in question and any Mogul-identified rings is also greater than 60 degrees, then that ring is considered an outlier. The outliers are highlighted in purple. The color gray indicates Mogul did not find sufficient equivalents in the CSD to analyse the geometry.

Bond lengths Bond angles
Torsions Rings

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry. 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. The following is a graphical depiction of the model fit to experimental electron density of all instances of the Ligand of Interest. In addition, ligands with molecular weight > 250 and outliers as shown on the geometry validation Tables will also be included. Each fit is shown from different orientation to approximate a three-dimensional view.

Mol Chain
Electron density around JN3 A 502: 2mF o -DF c (at 0.7 rmsd) in gray mF o -DF c (at 3 rmsd) in purple (negative) and green (positive)

Other polymers i ○
There are no such residues in this entry.