Biology subjects

Ridlon, J. M.

Publications and source records attributed to Ridlon, J. M..

13 recordsLinked to original sources

bioRxiv2026

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

Microbial steroid metabolism represents an underappreciated extension of the vertebrate endocrine system, with growing evidence that host-associated microbes contribute to the diversity and bioavailability of sex steroids within human tissues. Emerging studies have linked microbial androgen metabolism to urinary microbiome composition and to resistance to androgen deprivation therapy (ADT) in prostate cancer. While microbial pathways capable of converting steroid precursors such as cortisol to androgens, via the steroid-17,20-desmolase pathway, such as DesG-mediated interconversion of androstenedione to testosterone have been reported, the diversity of enzymes mediating downstream androgen interconversion remains incompletely defined. Here, we investigate the androgen-forming capabilities of anaerobic bacteria from the male genitourinary microbiome, focusing on NADPH-dependent 17{beta}-hydroxysteroid dehydrogenases (17{beta}-HSDHs) that catalyze interconversion of androstenedione and testosterone. We isolated androgen-forming bacterial strains from human male urine and identified a previously uncharacterized 17{beta}-HSDH encoded by Peptoniphilus obesi, demonstrated that this enzyme catalyzes the NADPH-dependent reduction of androstenedione to testosterone and the reverse oxidation reaction. Sequence similarity searches further identified a homologous 17{beta}-HSDH in Anaerococcus, which was synthesized and functionally validated, revealing conserved activity despite low sequence identity to the previously characterized urinary tract enzyme DesG. The enzymes were found to have broad substrate specificity for C19 and C18 17keto- and 17{beta}-hydroxysteroids. Together, these findings expand the known diversity of microbial 17{beta}-HSDHs and identify previously unrecognized androgen-forming activities within the genitourinary microbiome. ImportanceMicrobial steroid-transforming pathways may provide a mechanism by which commensal anaerobes contribute to androgen availability in the genitourinary tract. By identifying novel 17{beta}-hydroxysteroid dehydrogenases from Peptoniphilus and Anaerococcus, genera repeatedly associated with prostate cancer, this study provides mechanistic insight into how microbial steroid metabolism may influence hormone-driven disease.

microbiology↗

bioRxiv2025

The urinary pathobiont Actinobaculum massiliense generates androgens via the dirAB pathway

While overlooked during the Human Microbiome Project, characterizing the urinary microbiota in health and disease is a new frontier in microbiome science. Recent studies have associated differential abundance of bacterial taxa including Propionimicrobium lymphophilum and Actinobaculum/Actinotignum spp. with prostate cancer. In this study, we collected urine from subjects prior to prostate biopsy and applied a novel Human Sterolbiome Discovery High-throughput (HSDH) assay to identify culturable urinary bacteria with the ability to generate androgens. Application of the HSDH assay to urine samples led to the isolation of eight P. lymphophilum strains positive for cortisol side-chain cleavage (steroid-17,20-desmolase), 17{beta}-HSDH activity, or both. In addition, we isolated three strains of Actinobaculum massiliense that encode DHEA isomerase reductase (dir) genes. The dirA gene encodes a novel 3{beta}/17{beta}-hydroxysteroid dehydrogenase/{Delta}4,5-isomerase and the dirB gene encodes a novel 17{beta}-hydroxysteroid dehydrogenase isoform. Structural prediction and molecular dynamics reveal probable catalytic mechanisms based on the shared catalytic triad but distinct binding pocket geometries of the DirA and DirB that describe their respective reactions. Phylogenetic analysis of DirA and DirB revealed homologs in urinary tract commensals as well as bacteria associated with steroid degradation found in aquatic and terrestrial environments. Taken together, the development of the HSDH assay and the identification of the dir pathway genes is a significant advance in microbial endocrinology, laying the methodological foundation and providing the molecular basis for understanding the role of urinary tract bacteria in host endocrine physiology.

microbiology↗

bioRxiv2025

Characterization of an NADPH-dependent 17α-hydroxysteroid dehydrogenase encoded by the desF gene from the gut bacterium Clostridium scindens VPI 12708

Epitestosterone (epiT) is the isomer of the androgen testosterone. Historically, the role of epiT has remained unclear. Recently, it has been reported that epiT promotes AR-dependent prostate cancer cell proliferation. The gut bacterium Clostridium scindens VPI 12708 converts androstenedione (AD) to epiT. The bacterial enzymatic pathways involved in epiT formation have been reported, where the desF gene that encodes 17-hydroxysteroid dehydrogenase converts AD to epiT using NADPH as a cofactor. In this study, we quantitatively characterized DesF kinetic parameters and substrate specificity. The results revealed that the optimal pH for the reductive reaction is 7.0, and for the oxidative reaction it is 7.5 and 8.0. The kinetic analysis showed that for the reductive reaction, the KM was 8.67 {+/-} 2.04 {micro}M and the Vmax was 1.95 {+/-} 0.11 {micro}M min-1; for the oxidative direction, the KM was 27.17 {+/-} 3.56 {micro}M and the Vmax was 2.18 {+/-} 0.08 {micro}M min-1. Moreover, the substrate specificity analysis revealed that 11-keto-AD is the most favourable substrate for DesF, and the 17-keto group of 11-keto-AD can be converted to the 17-hydroxy group. These results are a significant advance in understanding epiT formation by the gut microbiome.

biochemistry↗

bioRxiv2024

Pangenome analysis of Clostridium scindens: a collection of diverse bile acid and steroid metabolizing commensal gut bacterial strains

Clostridium scindens is a commensal gut bacterium capable of forming the secondary bile acids deoxycholic acid and lithocholic acid from the primary bile acids cholic acid and chenodeoxycholic acid, respectively, as well as converting glucocorticoids to androgens. Historically, only two strains, C. scindens ATCC 35704 and C. scindens VPI 12708, have been characterized in vitro and in vivo to any significant extent. The formation of secondary bile acids is important in maintaining normal gastrointestinal function, in regulating the structure of the gut microbiome, in the etiology of such diseases such as cancers of the GI tract, and in the prevention of Clostridium difficile infection. We therefore wanted to determine the pangenome of 34 cultured strains of C. scindens and a set of 200 metagenome-assembled genomes (MAGs) to understand the variability among strains. The results indicate that the 34 strains of C. scindens have an open pangenome with 12,720 orthologous gene groups, and a core genome with 1,630 gene families, in addition to 7,051 and 4,039 gene families in the accessory and unique (i.e., strain-exclusive) genomes, respectively. The core genome contains 39% of the proteins with predicted metabolic function, and, in the unique genome, the function of storage and processing of information prevails, with 34% of the proteins being in that category. The pangenome profile including the MAGs also proved to be open. The presence of bile acid inducible (bai) and steroid-17,20-desmolase (des) genes was identified among groups of strains. The analysis reveals that C. scindens strains are distributed into two clades, indicating the possible onset of C. scindens separation into two species, confirmed by gene content, phylogenomic, and average nucleotide identity (ANI) analyses. This study provides insight into the structure and function of the C. scindens pangenome, offering a genetic foundation of significance for many aspects of research on the intestinal microbiota and bile acid metabolism.

microbiology↗

bioRxiv2024

Clostridium scindens: an endocrine keystone species in the mammalian gut

Clostridium scindens is a keystone human gut microbial taxonomic group that, while low in abundance, has a disproportionate effect on bile acid and steroid metabolism in the mammalian gut. 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-Bjorkhem 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 bile acid and steroid metabolism by strains of C. scindens, and the causal role of these pathways in host physiology and disease.

microbiology↗

bioRxiv2024

A widespread hydrogenase drives fermentative growth of gut bacteria in healthy people

Molecular hydrogen (H2) is among the most central, but least understood, metabolites in the human gastrointestinal tract (gut). H2 gas is produced in large quantities during bacterial fermentation and consumed as an energy source by bacteria and archaea. Disruption of H2 cycling is linked to gastrointestinal disorders, infections, and cancers, with H2 used as an indicator of gut dysfunction through breath tests. Despite this, the microorganisms, pathways, and enzymes mediating H2 production remain unresolved. Here we show that a previously uncharacterised enzyme, the group B [FeFe]-hydrogenase, drives most fermentative H2 production in the human gut. Analysis of stool, biopsy, and isolate (meta)genomes and (meta)transcriptomes show this hydrogenase is encoded by most gut bacteria and is highly expressed. Through analysis of 19 taxonomically diverse gut isolates, the group B [FeFe]-hydrogenase produces large amounts of H2 gas and supports fermentative growth of both Bacteroidetes and Firmicutes. Bacteroides particularly dominate H2 production. Biochemical and spectroscopic characterisation shows purified group B [FeFe]-hydrogenases are catalytically active and bind a di-iron active site. These hydrogenases are highly enriched in the guts of healthy individuals, but significantly depleted in favour of other fermentative hydrogenases in Crohns disease. Furthermore, we show that metabolically flexible respiratory bacteria are the most abundant H2 oxidizers in the gut, not sulfate reducers, methanogens, and acetogens as previously thought. This combination of enzymatic, cellular, and ecosystem-level analysis provides the first detailed understanding of H2 cycling in the human gut and reveals new links between microbiota function and gastrointestinal health.

microbiology↗

bioRxiv2024

An expanded metabolic pathway for androgen production by host-associated bacteria

A growing body of literature implicates host-associated microbiota in the modulation of circulating androgen levels in the host, which could have far-reaching implications for androgen-mediated diseases. However, the microbial genetic pathways involved in androgen production remain unknown. Here, we report the first host-associated microbial gene (desF) encoding an enzyme that catalyzes conversion of androstenedione to epitestosterone (epiT) in the gut bacterium, Clostridium scindens. Despite current dogma that epiT is a nuclear androgen-receptor (AR) antagonist, we demonstrate that epiT is a potent androgen, as assessed by its ability to promote prostate cancer cell growth and expression of prostate specific antigen (PSA). We then quantified the desF gene in fecal samples collected from individuals with advanced prostate cancer (rising blood PSA) undergoing androgen deprivation therapy combined with abiraterone acetate and prednisone (AA/P). Strikingly, fecal desF levels were elevated in a subset of individuals progressing on AA/P versus samples taken during AA/P response (stable). Importantly, we observed that AA does not inhibit the bacterial desmolase enzyme that is analogous to the human drug target of AA. We then determined that bacterial isolates from urine or prostatectomy tissue are capable of androgen production. From these isolates we detected 17{beta}-hydroxysteroid dehydrogenase (17{beta}-HSDH) activity, which has not been previously reported in urinary tract bacteria, and discovered the desG gene in urinary isolates encoding 17{beta}-HSDH that catalyzed conversion of androstenedione to testosterone. Applying advanced artificial intelligence and molecular dynamics, we predict the structures and ligand binding to DesF and DesG. Using a novel bioengineered microencapsulation technique, we demonstrate that urinary androgen-producing bacterial strains can also promote prostate cancer cell growth through steroid metabolism. Taken together, our results are a significant advance for steroid microbiology in humans and suggest that these microbial biotransformations should be further studied in the context of androgen-mediated physiological processes and diseases.

microbiology↗

bioRxiv2022

Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-trans orientation in which the sterol rings are "kinked", as well as small quantities of A/B-cis oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-{Delta}4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals. SIGNIFICANCE STATEMENTBile acid synthesis by vertebrates is central to digestion and nutrient signaling. Gut bacteria have evolved enzymes capable of converting primary bile acids to hundreds of secondary bile acids. While bile acid microbiology has been focused on the metabolism of ring hydroxyl groups and the carboxylated side-chain, very little is known about how bacteria alter the shape of the steroid ring system. Here, we describe enzymes expressed by Firmicutes that convert the "kinked" primary bile acid into "flat" secondary bile acids. Decades of research indicate that increased levels of secondary bile acids are risk factors for colorectal cancer. Hidden Markov Models developed from the BaiP and BaiJ enzyme sequences revealed significant enrichment in metagenomes of subjects with colorectal cancer.

biochemistry↗

bioRxiv2021

Diversity and distribution of sulfur metabolism in the human gut microbiome and its association with colorectal cancer

Microbial sulfidogenesis produces genotoxic hydrogen sulfide (H2S) in the human gut using inorganic (sulfate) and organic (taurine/cysteine/methionine) substrates, however the majority of studies have focused on sulfate reduction using dissimilatory sulfite reductases (Dsr). Recent evidence implicates microbial sulfidogenesis as a potential trigger of colorectal cancer (CRC), highlighting the need for comprehensive knowledge of sulfur metabolism within the human gut. Here we show that microbial sulfur metabolism is more abundant and diverse than previously studied and is statistically associated with CRC. Using ~17,000 bacterial genomes from publicly available stool metagenomes, we studied the diversity of sulfur metabolic genes in 667 participants across different health statuses: healthy, adenoma, and carcinoma. Sulfidogenic genes were harbored by 142 bacterial genera and both organic and inorganic sulfidogenic genes were associated with carcinoma. Significantly, anaerobic sulfite reductases were twice as abundant as dsr. We identified twelve potential pathways for reductive taurine metabolism including novel pathways, and prevalence of organic sulfur metabolic genes indicate these substrates may be the most abundant source of microbially derived H2S. Our findings significantly expand knowledge of microbial sulfur metabolism in the human gut, and highlight key gaps that limit understanding of its potential contributions to the pathogenesis of CRC.

microbiology↗

bioRxiv2020

Fxr signaling and microbial metabolism of bile salts in the zebrafish intestine

Bile salt synthesis, secretion into the intestinal lumen, and resorption in the ileum occurs in all vertebrate classes. In mammals, bile salt composition is determined by host and microbial enzymes, affecting signaling through the bile salt-binding transcription factor Farnesoid X receptor (Fxr). However, these processes in other vertebrate classes remain poorly understood. We show that key components of hepatic bile salt synthesis and ileal transport pathways are conserved and under control of Fxr in zebrafish. Zebrafish bile salts consist primarily of a C27 bile alcohol and a C24 bile acid which undergo multiple microbial modifications including bile acid deconjugation that augments Fxr activity. Using single-cell RNA sequencing, we provide a cellular atlas of the zebrafish intestinal epithelium and uncover roles for Fxr in transcriptional and differentiation programs in ileal and other cell types. These results establish zebrafish as a non-mammalian vertebrate model for studying bile salt metabolism and Fxr signaling.

physiology↗

bioRxiv2020

Identification and characterization of a gene encoding NADP(H)-dependent bile acid 12β-hydroxysteroid dehydrogenase from Clostridium paraputrificum ATCC 25780

Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with -orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the -orientation through an oxo-intermediate to the {beta}-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio-(C-3 vs. C-7) and stereospecific ( vs. {beta}) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso-(7-HSDH & 7{beta}-HSDH) and iso-(3-HSDH & 3{beta}-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12{beta}-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12{beta}-bile acid denoted epiDCA; however, gene(s) encoding this enzyme have yet to be identified. We confirmed 12{beta}-HSDH activity in cultures of Clostridium paraputrificum ATCC 25780. From six candidate C. paraputrificum ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12{beta}-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12{beta}-HSDH, leading to validation of two additional bile acid 12{beta}-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12-HSDH, we identified the first potential C-12 epimerizing strains: Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12{beta}-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.

microbiology↗

bioRxiv2020

Bacteria on steroids: the enzymatic mechanism of an NADH-dependent dehydrogenase that regulates the conversion of cortisol to androgen in the gut microbiome

Microorganisms extensively modify host steroids, but whether these reactions merely eliminate hormones or create signals with new biological identities is largely unknown. Gut bacteria have been known for more than four decades to reduce cortisol to 20-dihydrocortisol, yet the physiological consequence of this transformation remained unresolved. Here we show that microbial cortisol reduction creates a distinct host signalling molecule. A 2.0-[A] structure of the bacterial enzyme DesC, together with molecular dynamics, biochemical perturbation and hybrid quantum mechanics/molecular mechanics simulations, defines substrate recognition and an ordered hydride-transfer and proton-relay mechanism. In gnotobiotic mice, isogenic bacteria expressing active DesC--but not a catalytically inactive S47A variant produced 20-dihydrocortisol in the intestine and circulation and reprogrammed colonic transcription. In primary intestinal epithelial cells, 20-dihydrocortisol, but not cortisol, activated ERK-dependent inflammatory and growth-associated programmes. These responses required nuclear receptor subfamily 4 group A member 3 (NR4A3), whose purified ligand-binding domain bound 20-dihydrocortisol but showed no detectable binding to cortisol. A Chicago colonoscopy cohort linked chronic cortisol exposure, faecal desC, the microbial metabolite and colorectal phenotypes. Thus, bacterial metabolism can change receptor selectivity and biological activity rather than simply terminate host hormone action, expanding the endocrine chemistry of the host.

microbiology↗

bioRxiv2020

Infection with novel Bacteroides phage BV01 alters host transcriptome and bile acid metabolism in a common human gut microbe

The bacterial genus Bacteroides is among the most abundant and common taxa in the human gut, yet little is known about the phages infecting the group. Bacteroides phage BV01 (BV01) was identified as a prophage integrated on the chromosome of its host, Bacteroides vulgatus ATCC 8482. Phage BV01 is actively produced, and infects susceptible B. vulgatus hosts in the mouse gut. Infection with BV01 causes a generalized repression of the B. vulgatus transcriptome, downregulating 103 transcripts and upregulating only 12. Integration of BV01 disrupts the promoter sequence of a downstream gene encoding a putative tryptophan-rich sensory protein (tspO). Deletion of tspO and subsequent RNAseq analysis revealed that more than half of the differentially-regulated transcripts are shared with the BV01 lysogen, suggesting the transcriptomic response to BV01 is linked to tspO. Among these differentially-regulated transcripts are two encoding bile salt hydrolases. Bile acid deconjugation assays show that BV01 represses its hosts ability to hydrolyze bile acids in a tspO-dependent manner. Analysis of 256 published healthy human gut metagenomes suggests that phage integration adjacent to B. vulgatus-like tspO genes is rare within an individual, but common among humans. Finally, this work proposes a novel phage family that includes BV01, the Salyersviridae, whose host range spans the Bacteroides and is detectable in human-associated samples. Together, these findings highlight the importance of phage-host interactions to our understanding of how gut microbes sense and interact with their environment. IMPORTANCEThe links between human disease and the gut microbiome are numerous. Most mechanisms by which most gut microbes and their activities change and impact human health remain elusive. Phages, viruses that infect bacteria, are hypothesized to play a central role in modulating both community dynamics and functional activities. Here we have characterized an active prophage, BV01, which infects a pervasive and abundant human gut-associated species. BV01 infection alters its hosts transcriptional profile including its metabolism of bile acids, molecules implicated in mediating health and disease states in the gut. This highlights that prophages and other components of the variable genome should not be overlooked in bacterial genomes because they may dramatically alter host phenotypes. Furthermore, BV01 represents a new family of phages infecting human gut symbionts, providing a foundation for future investigations of phage-host interactions in these clinically-relevant but underexplored hosts.

microbiology↗