The Urinary Tract Commensal Peptoniphilus spp. Encodes a Novel 17β-Hydroxysteroid Dehydrogenase

This study identifies and characterizes novel NADPH-dependent 17β-hydroxysteroid dehydrogenases in the urinary tract commensals *Peptoniphilus obesi* and *Anaerococcus*, demonstrating their ability to interconvert androstenedione and testosterone and suggesting a previously unrecognized microbial mechanism for androgen availability in the genitourinary tract that may influence prostate cancer.

The Gist

Imagine your body is a bustling city. For a long time, scientists thought the "power plants" that generate your hormones (like testosterone) were located only inside your own factories (your adrenal glands and testes). But this new research reveals that there are hidden, tiny subcontractors living in your city's plumbing system (your urinary tract) that can also build these hormones, and they might be changing how diseases like prostate cancer behave.

Here is the story of that discovery, broken down into simple concepts:

1. The Hidden Subcontractors

Think of your urinary tract as a busy neighborhood. For a long time, we thought the bacteria living there were just harmless neighbors, like people walking their dogs. But this study found that some of these bacteria are actually chemical engineers.

Specifically, the researchers found two types of bacteria, Peptoniphilus and Anaerococcus, that are like little factories. They have been spotted more often in the neighborhoods of men with prostate cancer, but until now, no one knew exactly what "work" they were doing there.

2. The Magic Transformation (The Enzyme)

Inside these bacteria, there is a special tool called an enzyme. You can think of an enzyme as a molecular 3D printer or a shape-shifter.

• The Job: These bacteria take a "raw material" steroid called Androstenedione (which is a weak, inactive hormone precursor) and use their 3D printer to snap a few atoms into place, turning it into Testosterone (the powerful, active male hormone).
• The Reverse: They can also do the opposite! If there is too much testosterone, they can break it back down into the raw material. It's like a reversible elevator that can go up or down depending on what the bacteria need.

The researchers discovered that the bacteria use a specific "printer" (a protein they named after the bacteria) to do this. It's a brand-new model of printer that looks different from the ones we knew about before, but it does the same job.

3. Why This Matters for Prostate Cancer

Prostate cancer is like a weed that loves to drink testosterone. The standard treatment for this cancer is Androgen Deprivation Therapy (ADT). Think of ADT as cutting off the water supply to the weed; you stop the body from making testosterone so the cancer starves and shrinks.

Here is the problem:
If these bacteria in the urinary tract are acting as little factories, they can sneakily manufacture their own testosterone right next to the cancer, even when the doctor has cut off the main water supply. It's like the weed finding a hidden underground pipe that the doctors didn't know about. This could explain why some patients stop responding to treatment—the bacteria are keeping the cancer fed.

4. The "Universal Adapter"

The researchers tested these bacterial enzymes on many different types of steroids, not just the main ones. They found that these enzymes are like universal adapters. They can plug into and transform a wide variety of chemical shapes, including some that come from common medicines (like prednisone, a steroid drug).

This means these bacteria might be able to take drugs meant to treat other conditions and accidentally turn them into fuel for prostate cancer. It's a bit like a chef who, while trying to make a salad, accidentally turns the ingredients into a steak that feeds the cancer.

5. The Big Picture

This study is like finding a new map of the city's plumbing.

• Before: We thought the only source of testosterone was the body's main factories.
• Now: We know there are hidden bacterial factories in the urinary tract that can also make testosterone.

The Takeaway:
This doesn't mean the bacteria are "bad guys" in every situation; they are just doing what bacteria do—metabolizing chemicals. However, in the context of prostate cancer, their activity might be helping the disease grow.

By identifying exactly which tools (enzymes) these bacteria use, scientists can now start looking for ways to block those specific tools. Instead of just trying to stop the body from making hormones, doctors might one day be able to shut down these bacterial factories, cutting off the cancer's last line of fuel.

In short: Your body's plumbing has hidden factories that can build hormones. If you have prostate cancer, these factories might be feeding the disease. Now that we know how they work, we might be able to turn them off.

Technical Summary

1. Problem Statement

Microbial steroid metabolism is an emerging field suggesting that host-associated bacteria significantly influence the bioavailability and diversity of sex steroids, a concept termed the "microgenderome." While pathways for converting glucocorticoids (like cortisol) into androgens via steroid-17,20-desmolase (e.g., DesG in Propionimicrobium lymphophilum) have been identified, the diversity of enzymes responsible for downstream androgen interconversion remains poorly defined.

Specifically, there is a knowledge gap regarding:

• The specific microbial enzymes in the genitourinary microbiome that catalyze the interconversion of androstenedione (AD) and testosterone (T).
• The functional capabilities of Peptoniphilus and Anaerococcus genera, which are frequently associated with prostate cancer but have not previously been linked to steroid metabolism.
• Whether these bacteria can contribute to androgen availability in the prostate, potentially influencing resistance to Androgen Deprivation Therapy (ADT) in prostate cancer patients.

2. Methodology

The study employed a multi-disciplinary approach combining clinical microbiology, genomics, biochemistry, and phylogenetics:

• Sample Collection & Culturomics: Urine samples were collected from male patients (prostate cancer diagnosis and healthy controls) using strict "clean catch" protocols. Samples were cultured anaerobically on blood, Columbia, and Schaedler agar.
• Screening: Colonies were screened in PYG broth supplemented with 11-deoxycortisol (to detect side-chain cleavage) and 11β-hydroxyandrostenedione (11OHAD) to detect 17β-hydroxysteroid dehydrogenase (17β-HSDH) activity (conversion to 11-hydroxytestosterone).
• Genomics: Positive isolates underwent Whole Genome Sequencing (WGS) using PacBio Revio (long-read sequencing) to generate high-quality draft genomes. Peptoniphilus obesi strain CFH08 and Peptoniphilus harei strain CFH36 were identified.
• Gene Identification & Synthesis: Bioinformatic analysis identified a putative 17β-HSDH gene (CKIMDCCC_00407) in P. obesi. A homologous gene (GAN53_RS08700) was identified in Anaerococcus sp. Marseille-P9784 via BLASTP and phylogenetic analysis. Both genes were synthesized, codon-optimized, and cloned into E. coli expression vectors.
• Protein Purification: Recombinant proteins (rCKIMDCCC_00407 and rGAN53_RS08700) were expressed in E. coli BL21-CodonPlus and purified using affinity chromatography (Strep-tag and His-tag).
• Enzymatic Characterization:
  • pH Optimization: Activity was tested across pH 5.0–10.0.
  • Kinetics: Michaelis-Menten kinetics were determined for rCKIMDCCC_00407. Due to sigmoidal behavior, the Hill equation was used for rGAN53_RS08700.
  • Substrate Specificity: Enzymes were tested against a panel of C19 and C18 steroids (including 11-oxygenated androgens, DHEA, and estrogens) using spectrophotometry (NADPH/NADP+ consumption) and LC-MS for product verification.

3. Key Contributions

• Discovery of Novel Enzymes: Identification and biochemical validation of two novel NADPH-dependent 17β-HSDHs:
  1. rCKIMDCCC_00407 from Peptoniphilus obesi.
  2. rGAN53_RS08700 from Anaerococcus sp.
• Phylogenetic Expansion: Demonstration that these enzymes belong to a distinct clade within the Short-Chain Dehydrogenase/Reductase (SDR) family, sharing low sequence identity (~42%) with the previously characterized DesG enzyme but retaining similar catalytic functions.
• Functional Link to Prostate Cancer: Establishing that Peptoniphilus and Anaerococcus, genera consistently associated with prostate cancer in microbiome studies, possess the enzymatic machinery to generate testosterone from androstenedione.

4. Key Results

A. Isolation and Genomics

• P. obesi CFH08 (from a healthy control) and P. harei CFH36 (from a prostate cancer patient) were isolated.
• Genomic analysis of P. obesi revealed a 1.94 Mb genome with a predicted 17β-HSDH gene (CKIMDCCC_00407) annotated as a "3β-hydroxycholanate dehydrogenase," which was functionally re-annotated as an androgen-interconverting enzyme.

B. Enzymatic Activity of rCKIMDCCC_00407 (P. obesi)

• Bidirectional Catalysis: The enzyme catalyzes the NADPH-dependent reduction of androstenedione (AD) to testosterone (T) and the reverse oxidation.
• Kinetics (Reductive): $K_M$ = 14.4 µM, $k_{cat}$ = 75.9 s⁻¹, $k_{cat}/K_M$ = 5.3 s⁻¹·µM⁻¹. Optimal pH: 7.5.
• Kinetics (Oxidative): $K_M$ = 39.6 µM, $k_{cat}$ = 104.5 s⁻¹. Optimal pH: 9.0.
• Substrate Specificity: Broad specificity for 17-keto steroids.
  • Reductive preference: 11-ketoandrostenedione (11KAD) > 1,4-11KAD > AD > 5α-AD > 11OHAD > DHEA.
  • Oxidative preference: Testosterone > DHT > 11OHT.
  • Notably, estrone (E1) showed very low activity.

C. Enzymatic Activity of rGAN53_RS08700 (Anaerococcus)

• Sequence Identity: Shares 86% amino acid identity with the P. obesi enzyme.
• Kinetics: Exhibits sigmoidal kinetics (cooperative binding).
  • Reductive: $K_{0.5}$ = 23.32 µM, Hill coefficient = 4.65. Optimal pH: 6.0.
  • Oxidative: $K_{0.5}$ = 27.4 µM, Hill coefficient = 3.2. Optimal pH: 10.0.
• Substrate Specificity: Similar broad preference for 11-oxygenated steroids.
  • Reductive preference: 1,4-11KAD > 11KAD > 11OHAD > AD.
  • Oxidative preference: 11-ketotestosterone (11KT) > 11KT > T.
  • Unlike the P. obesi enzyme, rGAN53_RS08700 retained measurable activity toward estrogen substrates (E2).

D. Phylogenetic Analysis

• The enzymes cluster within the Bacillota phylum, specifically among Peptoniphilus and Anaerococcus species, as well as other urinary and gut commensals (e.g., Urinococcus, Collinsella). This suggests the trait is widespread among anaerobic commensals.

5. Significance

• Mechanistic Insight into Prostate Cancer: The study provides a molecular mechanism explaining how commensal anaerobes in the urinary tract could contribute to local androgen availability. Since Peptoniphilus and Anaerococcus are enriched in prostate cancer patients, their ability to convert AD to T may fuel tumor growth or contribute to resistance against ADT.
• Therapeutic Implications: Current ADT strategies (e.g., abiraterone) target host enzymes (CYP17A1) but do not inhibit bacterial steroid-17,20-desmolase or these newly discovered 17β-HSDHs. The existence of multiple, distinct microbial isoforms with varying substrate specificities (particularly for 11-oxygenated steroids derived from drug metabolism) suggests that microbial steroid metabolism is a significant, unaddressed factor in hormone-driven diseases.
• Expansion of the Sterolbiome: This work expands the known "sterolbiome" by identifying new enzymatic pathways in the genitourinary tract, highlighting that microbial steroid transformation is not limited to the gut but is a shared functional trait across multiple body habitats.
• Future Directions: The findings suggest that targeting these specific microbial enzymes or modulating the urinary microbiome could be a novel therapeutic strategy for castration-resistant prostate cancer.