Fig 1.
A) Bile acids are synthesized in the liver and secreted into the GI at a concentration of nearly 10 mM. During GI transit, approximately 90% is actively reabsorbed and recycled into the liver to be used in new rounds of digestion. During this recycling, bile acids signal through FXR, FGF19/15 and FGFR4 to regulate the rate limiting step of bile acid synthesis, CYP7A1. B) Bile acids are synthesized in two pathways. In the alternative pathway, only CDCA (chenodeoxycholate) derivatives (in humans) and CDCA / MCA (muricholate) derivatives (in mice) are made. In the classical pathway, both CDCA and CA (cholate) derivatives are formed. CA synthesis is dependent on CYP8B1 and the absence of CYP8B1 leads to only CDCA (and MCA) formation.
Fig 2.
Monoassociation of germ-free mice with bile acid metabolizing bacteria protects against CDI.
Monoassociation of germ-free mice with (A) and (B) C. hiranonis 10542 (N = 6), (C) and (D) C. leptum ATCC29065 (N = 6) or (E) and (F) C. scindens VPI12708 (N = 11) protects mice against CDI. Black line denominates monoassociated mice, red line denominates germ-free mice. On days 6 and 7 post CDI, monoassociated mice were given a single i.p. dose of clindamycin to induce recurrence [39]. A, C and E represent the average daily weights of the infected mice. B, D and F represent the Kaplan-Meier survival.
Table 1.
Bile acid profiles of conventionally raised wildtype, CYP8B1 heterozygous and CYP8B1 homozygous mice.
Fig 3.
Microbiome analyses of Cyp8b1+/- and Cyp8b1-/- mice.
A) Alpha-diversity analysis with Shannon index of fecal microbiome of heterozygous(N = 5) (HTZ) and homozygous (HMZ) (N = 5) mice treated with or without cefoperazone (CPZ)(N = 5); two tailed Mann-Whitney test was used for two-group comparison; B) Taxonomic abundance at family rank for mouse fecal microbiome; C) Beta-diversity analysis with Bray-Curtis dissimilarity distance metric and non-metric multidimensional scaling (NMDS); non-parametric statistical test for group comparisons was conducted with analysis of similarities (ANOSIM) method; D) Principal component analysis for PICRUSt2-based metabolic pathway abundance inferred from 16S amplicon data.
Fig 4.
Cholic acid derivatives are not required to initiate C. difficile infection.
A) Germ-free wild type (N = 3), CYP8B1 +/- (N = 3) or CYP8B1 -/- (N = 3)were infected with 105 C. difficile VPI10463 spores and monitored for signs of disease. Weight loss (B) or Kaplan-Meier survival plot (C) of CYP8B1 -/- mice infected with wildtype C. difficile UK1 or C. difficile JSC10 (cspC::ermB).
Fig 5.
Cholic acid derivatives are not required for protection against CDI.
C. scindens-monoassociated CYP8B1 +/- (N = 3) and CYP8B1 -/- (N = 4) mice were infected with C. difficile VPI10463 and monitored for signs of disease.
Table 2.
Bile acid profiles of C. scindens colonized CYP8B1 heterozygous and CYP8B1 homozygous mice.
Fig 6.
Fecal microbial therapy protects mice independent of cholic acid class bile acids.
Germ-free Cyp8b1-/- given cecal contents derived from a germ-free donor (GFCC) (N = 3) or mice colonized with human stool donor #1 with (N = 3) or without GFCC (N = 3) and human stool donor #2 with (N = 2) or without GFCC (N = 1) were infected with 104 spores derived from the C. difficile VPI10463 strain. Weight loss (A) or Kaplan-Meier survival curve (B) or disease scores (C) are illustrated.
Fig 7.
Untargeted metabolomics implicates Stickland metabolism / metabolites in colonization resistance.
A) Cecal contents from the indicated mouse samples (number of samples in parentheses) were sent for untargeted metabolomics. The abundance of 5-aminovalerate (B), proline (C) and glycine (D) in human stools samples derived from hospitalized controls (HC; N = 23) and CDI positive patients (N = 29) is quantified. Two-tailed Mann-Whitney test was used for group comparison: NS, not significant; ** p < 0.01; *** p < 0.0001.
Fig 8.
Stickland metabolism is important for colonization resistance.
Spores derived from the C. difficile VPI10463 strain were inoculated into germ-free (N = 3) or mice monoassociated with (A) and (B) P. bifermentans (N = 11) or (C) and (D) A. hallii (N = 4). On day 6 post CDI, monoassociated mice were given a single i.p. dose of clindamycin to induce recurrence [39]. A and C represent the average weights of the infected mice. B and D represent the Kaplan-Meier survival.