Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

Abstract

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, (1)H and (13)C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (≈ 1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative "transketolase" (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic "rheostat" controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.

Keywords: RNA-Seq; microbiome; steroid.

Fig. 1.

HR-APCI-MS of steroid-17,20-desmolase reaction product.

Fig. 2.

The molecular structure of the steroid-17,20-desmolase reaction product identified as 11β-hydroxyandrosten-3,17-dione. An Oak Ridge Thermal Ellipsoid Plot (ORTEP) view of the molecule with atomic labeling (thermal ellipsoids are drawn at 50% probability) (See also supplementary Table II for crystallographic details and supplementary Table IV for atomic coordinates, bond angles, and bond lengths)Oxygen atoms are in red, carbon atoms in grey, and hydrogen atoms in white. A: Side view of the molecule. B: Top view of the molecule. C: Standard chemical formula of the molecule (top left), stereochemical formula of the molecule displaying ¹³C NMR shifts (ppm) (top right), and stereochemical formula of the molecule displaying ¹H NMR shifts (ppm) (bottom). See supplementary text and supplementary Table III for further description.

Fig. 3.

Measurement of cortisol induction, mRNA purification, and effect of cortisol induction on transcriptome of C. scindens ATCC 35704. A: Prior to isolation of mRNA for RNA-Seq, whole cells were measured for steroid-17,20-demolase activity by measurement of 11β-OHAD formation (n = 3). B: One microgram of total RNA was subjected to bead-capture hybridization (see Materials and Methods for details). We observed ∼80% removal of total RNA (Elution) by streptavidin bound magnetic beads coated with biotinylated oligonucleotides designed to bind 16s rRNA and 23s rRNA molecules. C: RNA gel demonstrating reduction in intensity of rRNA bands following bead-capture hybridization. Control (Ctrl) sample (10 μg total RNA) was subjected to bead-capture hybridization with beads lacking biotinylated capture oligos. Enrichment (Enrich) fraction containing enriched mRNA was sequenced. D: Results of RNA-Seq experiments comparing transcriptome data from control cells grown in BHI without cortisol and cortisol-induced cells. Upregulated genes are represented in blue, downregulated in red. Gene annotations from BLAST were used to group genes by general function (see supplementary Dataset I).

Fig. 4.

RNA-Seq data suggests a hypothesis for the mechanism of steroid-17,20-desmolase. We detected a cluster of four genes upregulated 1,000-fold by cortisol. This operon contains two genes encoding the N- and C-terminal subunits of a putative transketolase. The C. scindens ATCC 35704 genome contains additional transketolase genes which are not upregulated by cortisol and which display higher sequence identity with other transketolases found in nr BLASTP database (99–91% vs. 45–41% in first five hits; E-values 0.0 vs. 7e⁻⁷¹ to 4e⁻⁶⁰). A comparison of the structure of the side chain removed by steroid-17,20-demolase, and transketolation reactions in the pentose-phosphate pathway, suggests that steroid-17,20-desmolase may proceed by TPP-dependent transketolation. HD, haloacid dehalogenase.

Fig. 5.

SDS-PAGE and Western immunoblot of recombinant DesC. A: SDS-PAGE. B: Western blot. M, protein marker; 1, crude extract of E. coli BL21(DE3)RIL after expression of recombinant DesC-ST; 2, purified recombinant DesC-ST after Strep-Tactin affinity chromatography.

Fig. 6.

Maximum likelihood phylogenetic tree of desC. Values on nodes represent bootstrap support (only 50 or higher shown). A: Overall tree of 341 sequences from the second step of the analysis (see main text for details). B: Details of the region of the tree containing C. scindens’ desC [shaded area in panel (A)]. Colors reflect selected taxonomic groups, as depicted in the in-figure key. C: Organization of genes surrounding desC in shaded region of (B). Colors reflect gene function, as described in the in-figure key.

Fig. 7.

Cortisol metabolism by the human gut microbiome. Members of the human gut microbiome are capable of reducing, and epimerizing the 3-oxo group, reducing the Δ⁴-bond, oxidation/reduction of the 20-oxo group, removing the side-chain by steroid-17,20-desmolase, 21α-dehydroxylation, and epimerizing the 17-oxo group of 11β-OHAD.

Fig. 8.

Model for cellular function of steroid-17,20-desmolase. Functional characterization of the desC gene (yellow) product supports our hypothesis that the transketolase encoded by the desAB genes (red, orange) encode steroid-17,20-desmolase, which proceeds by transketolation. Transketolases are TPP-dependent, and we observe an upregulation of genes involved in TPP synthesis. The desD gene (green) is predicted to encode a cortisol transport protein, and an ABC-type transporter was identified (500-fold induction; blue) which could serve to pump 11β-hydroxyandrostenedione out of the cell. We also observed several pentose phosphate pathway genes upregulated, suggesting that the two-carbon side chain might enter the pentose phosphate pathway. We hypothesize that the 20α-HSDH encoded by the desC gene regulates flux of the two-carbon fragment into the pentose phosphate pathway, as 20α-hydroxycortisol is not a substrate for steroid-17,20-desmolase.