Bacterial steroid-17,20-desmolase is a taxonomically rare enzymatic pathway that converts prednisone to 1,4-androstanediene-3,11,17-trione, a metabolite that causes proliferation of prostate cancer cells

Abstract

The adrenal gland has traditionally been viewed as a source of "weak androgens"; however, emerging evidence indicates 11-oxy-androgens of adrenal origin are metabolized in peripheral tissues to potent androgens. Also emerging is the role of gut bacteria in the conversion of C₂₁ glucocorticoids to 11-oxygenated C₁₉ androgens. Clostridium scindens ATCC 35,704 is a gut microbe capable of converting cortisol into 11-oxy-androgens by cleaving the side-chain. The desA and desB genes encode steroid-17,20-desmolase. Our prior study indicated that the urinary tract bacterium, Propionimicrobium lymphophilum ACS-093-V-SCH5 encodes desAB and converts cortisol to 11β-hydroxyandrostenedione. We wanted to determine how widespread this function occurs in the human microbiome. Phylogenetic and sequence similarity network analyses indicated that the steroid-17,20-desmolase pathway is taxonomically rare and located in gut and urogenital microbiomes. Two microbes from each of these niches, C. scindens and Propionimicrobium lymphophilum, respectively, were screened for activity against endogenous (cortisol, cortisone, and allotetrahydrocortisol) and exogenous (prednisone, prednisolone, dexamethasone, and 9-fluorocortisol) glucocorticoids. LC/MS analysis showed that both microbes were able to side-chain cleave all glucocorticoids, forming 11-oxy-androgens. Pure recombinant DesAB from C. scindens showed the highest activity against prednisone, a commonly prescribed glucocorticoid. In addition, 0.1 nM 1,4-androstadiene-3,11,17-trione, bacterial side-chain cleavage product of prednisone, showed significant proliferation relative to vehicle in androgen-dependent growth LNCaP prostate cancer cells after 24 h (2.3 fold; P < 0.01) and 72 h (1.6 fold; P < 0.01). Taken together, DesAB-expressing microbes may be an overlooked source of androgens in the body, potentially contributing to various disease states, such as prostate cancer.

Keywords: Microbiome drug metabolism; Prednisone; Prostate cancer; Steroid desmolase.

Figure 1.. Large scale phylogenetic analysis of DesA.

A: Maximum-likelihood phylogenetic tree of 37,894 protein sequences from NCBI’s non-redundant database that were similar to C. scindens DesA and were between 200 and 800 amino acids in length. B: Phylogenetic analysis of solely the sequences present in the short-sequence clade (i.e., the subtree where the DesA protein is placed). C: Zoom into the part of the tree where C. scindens DesA is located; numbers on nodes are FastTree-calculated support values, with only values above 0.5 being shown.

Figure 2.. Thresholded sequence similarity networks represent sequences as nodes (circles) and pairwise sequence relationships (alignments) better than a set threshold as edges (lines).

As the threshold becomes more stringent, sequences break up from the main cluster. Nodes have been colored according to sequence length in amino acids (scale at the bottom). A: At threshold values below 50, proteins sequence are divided in two main clusters. B: At a threshold value of 75, the first group of sequences becomes independent from the main clusters. This group (red circle) contains proteins from the DesA family. C: At a threshold value of 100, there is a clear separation into multiple groups. Proteins from the DesA family remain clustered (red circle), and proteins from the DesB family are separated into their own cluster (cyan circle). D: At threshold values above 150, the majority of sequences break up into disconnected groups.

Figure 3.. In-depth view of the DesA and DesB groups and their genomic context.

A: Species of organisms identified within the functional clusters of family DesA and DesB. Proteins labeled as “multispecies” are derived from metagenomics data and, thus, don’t have a single species associated with them. B: Genomic context of proteins identified within the DesA and DesB functional clusters.

Figure 4.. Side-chain cleavage of endogenous and glucocorticoid drugs by whole cells of gut bacterium Clostridium scindens ATCC 35704 and urinary tract isolate Propionimicrobium lymphophilum ACS-093-V-SCH5.

A: Steroid-17,20-desmolase reaction showing endogenous (highlighted in gray) and exogenous (highlighted in blue) compounds as substrates. Anticipated product (loss of C₂O₂H₄) of side chain cleavage is 60.02 m/z smaller than substrate. B: LC/MS in single ion monitoring (SIM) mode of extracts from C. scindens incubated with 50 μM substrate. C: LC/MS in single ion monitoring (SIM) mode of extracts from P. lymphophilum incubated with 50 μM substrate. Chromatographs representative of three biological replicates.

Figure 5.. SDS-PAGE of purified recombinant DesA and DesB from Clostridium scindens ATCC 35704 and NADPH-dependent 17β-HSDH from Cochliobolus lunatus.

Affinity purified recombinant steroid-17,20-desmolase (rDesA and rDesB) co-expressed in pETDuet (Lane DesAB), affinity purified recombinant 17β-hydroxysteroid dehydrogenase expressed in pET21 (Lane 17β-HSDH), and molecular mass marker (Lane M).

Figure 6.. Total ion count chromatographs of rDesAB reaction products.

Reaction was initiated with 2 μM of pure rDesAB enzyme to 50 mM MOPS buffer (pH 7.0), 100 μM of substrate (blue) in the presence of 1 μM MnCl2 and 10 μM TPP at room temperature. After 2 minutes, while the reactions were stopped with the addition of 1/10 volume of 1 M HCl and vortexing to drop the pH below 2. A final concentration of 50 uM of internal standard, 20β-dihydrocortisol (green), was spiked into the reaction. Products formed were identical with respect to retention time and loss of 60 amu consistent with side-chain cleavage.

Figure 7.. Measurement of LNCaP cell proliferation in the presence of steroid-17,20-desmolase reaction products.

Induction of cell proliferation in androgen-dependent LNCaP cells by DHT (dihydrotestosterone), 11KA (11-keto-androstenedione), and AT (1,4-androstadiene-3,11,17-trione). Cells (2.5 × 10⁴ cells) were seeded in a 96-well plate in RPMI 1640 media containing 10% charcoal: dextran stripped FBS for 24 hours before adding 0.1 and 1 nM steroid. MTS assay was carried out 24 and 72 hours after. Results are shown as mean ± SEM of four independent experiments with three replicates each. Differences between the compound of interest and Vehicle were conducted by ANOVA, followed by Dunnett’s test (* = p<0.05, ** = p<0.01, and *** = p<0.001 relative to the vehicle control).

Figure 8.. Hypothetical model of physiological relevance of steroid-17,20-desmolase activity in urinary microbes in the context of prostate cancer.

Urinary microbes inhabiting a minimal nutrient environment metabolize glucocorticoids for energy, generating androgens that diffuse into surrounding prostate tissue. Microbe-derived androgens activate androgen receptor (AR), leading to increased proliferation of androgen-dependent prostate cancer cells and ultimately resulting in prostate cancer progression.