Characterization of glucocorticoid metabolism by microbial steroid-17,20-desmolase

Ly, Lindsey K

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https://hdl.handle.net/2142/116065 Copy

Description

Title

Characterization of glucocorticoid metabolism by microbial steroid-17,20-desmolase

Author(s)

Ly, Lindsey K

Issue Date

2022-07-12

Director of Research (if dissertation) or Advisor (if thesis)

Ridlon, Jason M

Doctoral Committee Chair(s)

Gaskins, Rex

Committee Member(s)

• Donovan, Sharon M
• Steelman, Andrew J

Department of Study

Nutritional Sciences

Discipline

Nutritional Sciences

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• glucocorticoid
• androgen
• microbiome
• steroid
• desmolase

Abstract

The mechanisms governing the gut microbiome serving as a virtual endocrine organ are poorly understood, but harbor immense potential as a therapeutic target. The gut microbiome generates steroid metabolites that act as hormones, affecting not only local physiology, but also having systemic consequences if absorbed into circulation. One microbial pathway of emerging interest is the steroid-17,20-desmolase (DesAB) pathway that converts cortisol, a C21 glucocorticoid (GC), to 11b-hydroxyandrostenedione (11b-OHAD), a C19 pro-androgen. This is of clinical significance because 11b-OHAD is a precursor to 11-oxy-androgens, a class of androgens with potent androgenic activity, which can be generated by both host and resident microbes. In this work, the number of desAB-encoding microbes was expanded using phylogenetic and sequence similarity network analyses. Additionally, the number of substrates for DesAB were expanded to also include pharmaceutical analogs of cortisol using both purified recombinantly expressed DesAB (rDesAB) and whole cells from two microbes from gut and urinary tract, Clostridium scindens ATCC 35704 and Propionimicrobium lymphophilum ACS-093-V-SCH5, respectively. LC/MS analysis showed that both microbes were able to side-chain cleave prednisone, prednisolone, 9-fluorocortisol, dexamethasone, allo-tetrahydrocortisol, cortisone, and cortisol. Interestingly, rDesAB showed highest activity against prednisone and prednisolone. In vitro culturing of androgen-responsive prostate cancer cells (LNCaP) showed that 1,4-androstadiene-3,11,17-trione, the product of bacterial side-chain cleavage of prednisone, caused significant proliferation relative to vehicle at 24 and 72 hours. We hypothesize that urinary microbes inhabiting a minimal nutrient environment metabolize GCs for energy, generating androgens that diffuse into surrounding prostate tissue, which may lead to increased proliferation of androgen-dependent prostate cancer cells. Phylogenetic analysis revealed that DesAB is a taxonomically rare pathway that evolved from sugar transketolases, however the reason for the evolution remains unknown. To better understand the physiological function of DesAB, the in vitro cortisol-induced transcriptome of a desAB-encoding gut microbe, Butyricicoccus desmolans ATCC 43058, was analyzed using RNA-Seq. Differential gene expression revealed that the DesAB pathway (desABED) was most upregulated (100-300-fold) in the presence of cortisol. Several candidate GC importers and androgen exporters were upregulated, which may act as important drug targets to mitigate 11b-OHAD production. For B. desmolans, we hypothesize that cortisol may be important for signaling upregulation of important gut colonization functions, including ferrous transporters and flagellar assembly. However, in vivo studies utilizing knockout mutants are needed to understand the effect of DesAB on both microbial and host physiologies. Until genetic systems are available to engineer desAB-knockout mutant strains, a soluble inactive rDesAB was generated using site directed mutagenesis on an active site residue (E70A), which can be integrated into the chromosome of E. coli for future mechanistic studies in vivo. Germ-free piglets are an excellent model to study the effect of DesAB in vivo as they are cortisol-dominant like humans. Germ-free piglets (n=3/group) were colonized with E. coli MG1655, E. coli MG1655 and C. scindens ATCC 35704 (with DesAB activity), or E. coli MG1655 and C. scindens BL389WT3D (lacking DesAB activity). Unfortunately, the feasibility of delivering germ-free piglets using a “closed” Cesarean-section using a surgical bubble was determined to be unreliable and impractical. As a significant Staphylococcus contamination was identified in all piglets (n=9) at the end of the study, further analyses were terminated. It is important to understand the contribution of bacterial androgen production in vivo because surface and nuclear AR are expressed in numerous tissues of the body, including colonocytes and cells of the innate and adaptive immune system. Understanding bacterial androgen production is particularly important when assessing risk and progression in diseases that are exacerbated by high androgen levels, such as prostate cancer or polycystic ovary syndrome. Basic understanding of why and how the microbes are responding to cortisol, will lead to rational modulation of the extracellular environment to potentially shift metabolism away from side-chain cleavage of GCs, which may have translating applications for human dietary interventions if DesAB is indeed contributing to disease.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Lindsey Ly

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