Clostridium scindens: history and current outlook for a keystone species in the mammalian gut involved in bile acid and steroid metabolism

Abstract

Clostridium scindens is a keystone bacterial species in the mammalian gut that, while low in abundance, has a significant impact on bile acid and steroid metabolism. Numerous studies indicate that the two most studied strains of C. scindens (i.e. ATCC 35704 and VPI 12708) are important for a myriad of physiological processes in the host. We focus on both historical and current microbiological and molecular biology work on the Hylemon-Björkhem pathway and the steroid-17,20-desmolase pathway that were first discovered in C. scindens. Our most recent analysis now calls into question whether strains currently defined as C. scindens represent two separate taxonomic groups. Future directions include developing genetic tools to further explore the physiological role of bile acid and steroid metabolism by strains of C. scindens and the causal role of these pathways in host physiology and disease.

Keywords: Clostridium scindens; 7α-dehydroxylation; Hylemon–Björkhem pathway; gut microbiome; secondary bile acids; steroids; sterolbiome.

Figure 1.

Investigators who worked on the side chain cleavage of steroids, dehydroxylation of bile acids by human fecal bacteria, and isolation and identification of the model gut bacteria C. scindens ATCC 35704 and VPI 12708.

Figure 2.

Timeline in the study of bacterial steroid-17,20-desmolase.

Figure 3.

Colony and cellular morphology of C. scindens ATCC 35704. (A and B) Electron micrographs of C. scindens ATCC 35704 (Bokkenheuser et al. 1984). Used with kind permission from Oxford University Press. Gram stain (C) of cells and colonies (D) of C. scindens ATCC 35704 grown on anaerobic EG agar after three days of incubation. Used with kind permission from RIKEN and the Japan Collection of Microorganisms.

Figure 4.

The current model of the Hylemon–Björkhem Pathway of bile acid 7α-dehydroxylation of cholic acid by C. scindens strains. Steps V-VIA(VIb) have been shown to be catalyzed by BaiH/BaiN and BaiJ enzymes (Funabashi et al. , Lee et al. , Meibom et al. 2024). *Step VIb–VIIb has been shown to be catalyzed by BaiJ and BaiP (Lee et al. , Meibom et al. 2024). Bile acid exporters (orange) have not yet been identified. Each enzymatic step is described in detail in the associated text. Modified from previously published work (Devendran et al. 2019).

Figure 5.

Gene organization of bile acid- and steroid-metabolizing genes in C. scindens VPI 12708 and ATCC 35704. The complete genome from each strain has been deposited previously (Devendran et al. , Olivos-Caicedo et al. 2023).

Figure 6.

Structure and catalytic mechanism of BaiE, the bile acid 7α-dehydratase. (A) Visual Molecular Dynamics (VMD) Model of BaiE, a potential drug target, and rate-limiting enzyme responsible for the formation of toxic and cancer-causing bile acids. Left: Ribbon diagram of Ligand-bound subunit (purple) is overlaid with apo-enzyme (cyan). Bile acid ligand displayed in red. Right: Monomeric space-filling subunit structure of BaiE from P. hiranonis (purple) with bile acid ligand (red). (B) Trimeric native form of BaiE from P. hiranonis with mixed ribbon and space-filling subunits. Ligand-bound subunit (purple) is overlaid with apo-enzyme (cyan). Bile acid ligand displayed in red. (C) Left: Probable productive binding mode of 3-oxo-Δ⁴-CDCA. Blue dashed lines and adjacent numbers are predicted interaction of His83 with C7-OH and C6 atoms and Y30-OH group with C3-oxo atom of 3-oxo-Δ⁴-CDCA. The 6α-H closest to H83 colored magenta, and 6β-H away from H83-Nε2 atom colored brown. Right: Predicted stacking interaction involving the adenine group of the coenzyme (CoA) moiety of 3-oxo-Δ⁴-CDCA∼SCoA with Y115. The key interaction of the bile acid moiety of the docked CoA-bile acid ester with the active site residues is like what is predicted in left panel. Carbon atoms of protein residues and product molecules are colored gold and green, respectively. H, O, N, P, and S atoms are colored gray, red, blue, orange, and olive, respectively. (D) Proposed mechanism of catalysis by BaiE. Y30 acts as a general acid protonating C3-oxyanion, stabilizing negative charge, and potentiating electron shift, destabilizing C6-6αH. H83, stabilized by D35, acts as a general base, executing deprotonation and ensuring protonation reaction with the subsequent release of water. Figure modified from previously published work (Bhowmik et al. 2016). Images in A and B courtesy of Prof. Rafael C. Bernardi, Auburn University.

Figure 7.

Bile acid oxidoreduction by C. scindens. While CA is known to be transported by BaiG, it is assumed that oxo-bile acids are also recognized by this transporter, but this has yet to be determined. The import of 3-oxo-CA (or 3-oxo-CDCA) is predicted to be ligated to coenzyme A by BaiB and funneled into the Hylemon–Björkhem (HB) pathway and converted to DCA. 7-oxo-DCA has been shown to be converted to CA by NADPH-dependent 7α-HSDH (Baron et al. 1991). CA is then ligated to coenzyme A by BaiB and oxidized to 3-oxo-CA∼SCoA by the BaiA (NADH-dependent 3α-HSDH) (Bhowmik et al. 2014). 12-oxo-LCA is converted to DCA by NADH-dependent 12α-HSDH (Doden et al. 2018).

Figure 8.

The steroid-17,20-desmolase pathway in host associated bacteria, including C. scindens. (A) 20α-dihydrocortisol is converted to cortisol by DesC (NADH-dependent 20α-HSDH) and cortisol to 11β-hydroxyandrostenedione by DesAB (steroid-17,20-desmolase) encoded by C. scindens ATCC 35704 (Ridlon et al. , Devendran et al. 2018). 20β-dihydrocortisol is not a substrate for C. scindens; however, organisms such as B. desmolans, C. cadavaris, and P. lymphophilum express DesE, an NADH-dependent 20β-HSDH (Devendran et al. 2017). 11β-hydroxyandrostenedione is converted by 17α-HSDH to 11β-hydroxy-epi-testosterone encoded by C. scindens VPI 12708 (de Prada et al. 1994). (B) Gene cluster desABCD encoding steroid-17,20-desmolase (DesAB), DesC (NADH-dependent 20α-HSDH), and a putative cortisol transport protein (DesD) in C. scindens ATCC 35704 (Ridlon et al. 2013). (C) The desA and desB genes encode predicted N-terminal and C-terminal transketolases. An analogous reaction is predicted between sugar transketolation and steroid-17,20-desmolase. (D) The host liver reduces cortisol to tetrahydrocortisol or allotetrahydrocortiol, some of which undergoes enterohepatic circulation via the bile. Clostridium scindens is capable of recognizing allotetrahydrocortisol, converting this to 11β-hydroxyandrosterone. We recently discovered that C. scindens VPI 12708 encodes the desF gene, which converts 17-keto androstanes to derivatives of epitestosterone. Epitestosterone and the 5α-reduced derivative of epiT (5α-dihydroepiT) have been shown to be an androgen receptor agonist (Schiffer et al. 2024). Modified from previously published work (Ly et al. 2021).

Figure 9.

The role of bile acid and tryptophan metabolism in germination and vegetative growth of C. difficile. Taurocholic acid is deconjugated, mainly in the large intestine, by diverse gut microbial taxa. Free cholic acid is imported into a few species of Bacillota that harbor the bai regulon. Direct Pathway: After several oxidative steps and rate-limiting 7α-dehydration, 3-oxo-Δ⁴-DCA becomes a substrate for BaiCD forming DCA or BaiP/BaiJ forming alloDCA. Indirect Pathway: DCA is imported into Bacteroidetes strains that express 3α-HSDH and 5β-reductase (5BR), which converts DCA to 3-oxo-Δ⁴-DCA. Expression of 5α-reductase (5AR) and 3β-HSDH sequentially reduce 3-oxo-Δ⁴-DCA to iso-allo-DCA. Allo-DCA generated by Bacillota is also isomerized to iso-allo-DCA via 3α-HSDH and 3β-HSDH expressing strains of E. lenta and other taxa. While TCA is a germination factor for C. difficile, DCA and isoalloLCA have been shown to be inhibitory toward C. difficile vegetative growth in vitro and in vivo. Secondary bile acids, including DCA and allo-DCA, are associated with increased risk of CRC. In addition, C. scindens strains have been shown to convert L-tryptophan to 1-acetyl-β-carboline, which promotes synergistic inhibition of C. difficile in the presence of hydrophobic secondary bile acids (Kang et al. 2019). Modified from a previously published work (Lee et al. 2022).

Figure 10.

Phylogenomics and diversity of bai and des genes in strains of C. scindens. The formation of two clades is shown, Clade 1 (green) includes 15 strains and Clade 2 (blue) 19 strains. Bootstrap support values above 50% are shown in yellow stars at nodes.

Figure 11.

Key metabolic pathways in the core genome of C. scindens (Olivos-Caicedo et al. 2025).

Figure 12.

A proposed model for the interaction between glucose fermentation, cortisol metabolism, and bile acid 7α-dehydroxylation by C. scindens ATCC 35704. EMP, Embden–Meyerhof–Parnas pathway.