STUDIES IN THE BIOTRANSFORMATION OF GLUCOCORTICOIDS TO ANDROSTANES BY GASTROINTESTINAL BACTERIA IN ANIMALS IMPORTANT TO AGRICULTURE AND CONSERVATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007734
• Project Start Date: Nov 12, 2015
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Animal Sciences

Non Technical Summary
Cortisol is released and circulates throughout the body during stress. Cortisol is released from the body in urine and feces. In the gastrointestinal tract, some anaerobic bacteria are capable of converting cortisol, a glucocorticoid into a variety of androstanes/androgens. These metabolites have the potential to alter reproductive success, and lead to disease states, particularly under prolonged stress. It is thus important to determine if this microbial metabolism leads to reduced product-quality (meat, milk, eggs), reduced reproductive success, or fitness in the case of conservation biology.The proposed studies will provide the basis for a mechanistic understanding of how bacteria convert cortisol to androstanes/androgens, identify microbial species inhabiting the gut of agriculturally-important animals capable of this metabolism, provide DNA sequence data on the genes encoding cortisol-metabolizing enzymes, and identify potential therapeutic targets aimed at inhibiting these bacteria.The ultimate goals of this research are to provide the knowledge base from which practical tools may be developed to allow researchers to detect and quantify these bacteria in order to determine if they correlate with any pathophysiological states in animals important to agriculture, conservation, and even medicine. These studies may thus warrant future mechanistic studies to determine how these bacterial metabolites play causal roles in pathophysiology. To come back full circle, the knowledge gained from studying two key bacterial enzymes, the cortisol transporter, and the enzyme catalyzing the conversion of cortisol to androstane, provide specific targets for drug development to inhibit bacterial metabolism of cortisol.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
303	3910	1080	100%

Knowledge Area
303 - Genetic Improvement of Animals;

Subject Of Investigation
3910 - Cross-commodity research--multiple animal species;

Field Of Science
1080 - Genetics;

Keywords

animal wellfare

stress measurement

glucocorticoids

androstanes

Goals / Objectives
Problem Statement 1: Stress is quantified in animals of agricultural importance, and conservation through fecal measurement of cortisol and its metabolites produced by gut microbial species; however, before DNA-based approaches can be applied to studying this gut microbial metabolism, the genes responsible must be identified and characterized.Major Goal 1: Objectives 1 and 2 seek to identify and characterize the genes encoding two important functions in microbial metabolism of cortisol. Objective 1 will characterize the gene(s) responsible for converting cortisol to 11β-hydroxyandrostenedione. Objective 2 will characterize the gene that transports cortisol into the bacterial cell.Major Goal 2: Because DNA sequences for a particular gene vary between strains of the same species, and even moreso between different species and genera of bacteria, isolation of bacteria with cortisol-inducible genes and DNA sequencing of these genes is an important goal of this research. By comparing these sequences, PI Ridlon is confident that regions can be identified in these gene sequences from which quantitative assays can be developed to identify and quantify these bacteria using DNA-based approaches.Major Goal 3: The basic science knowledge of the transport of cortisol (desD), and its biotransformation from cortisol (C21) to androstrane (C19) provide two potential drug-targets to specifically inhibit this activity if future work demonstrates a causal role in pathophysiology.PI Ridlon has a strong background and publication record in recombinant DNA technology and enzyme characterization. PI Ridlon also has over a decade experience working with radioactively labeled steroid molecules and their use in working out metabolic pathways in gut bacteria. Furthermore, PI Ridlon has extensive experience locating and characterizing genes both at the bench, and using computational analysis. Therefore, PI Ridlon is confident these goals will be achieved within the requested time frame.Problem Statement 2: Organisms capable of encoding cortisol metabolism have only been isolated from human feces, sewage, and domesticated cat feces. It will therefore be important to isolate and characterize additional strains from chicken, sheep, pigs, and cows.Major Goal 4: PI Ridlon will apply enrichment-based anaerobic microbiology techniques coupled with analytical radio-labeled cortisol conversion assays to isolate gut microbes capable of cortisol-metabolism from feces collected from chicken, sheep, pigs, and cows.PI Ridlon has over a decade experience cultivating anaerobic bacteria, including isolating bacteria from fecal samples. Therefore, PI Ridlon anticipates success of this goal during the time period requested.

Project Methods
The first two objectives will involve recombinant DNA technology involving the overexpression of protein in Escherichia coli from a gene found in Clostridium scindens, a gut bacterium. Objective 1 will involve affinity chromatography which will allow purification of recombinant protein with an engineered streptavidin affinity tag on a resin linked with biotin, the ligand of streptavidin. Purified recombinant proteins will then be studied mechanistically in order to obtain kinetic constants that will inform researchers about the reaction rate, binding order of substrates, and substrate recognition. Analytical methods to separate steroid reactants and products such as thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) against known standards will be utilized. Objective 2 will measure the rate by which [4-14C] cortisol is transported into E. coli overexpressing the recombinant streptavidin-tagged desD gene product relative to a control containing the expression vector (pSport-1) without desD insert. Thus, cells will be collected, filtered and quantified by liquid scintillation spectrometry to determine kinetic constants. Mechanistic study of the transport protein will be determined by use of ionophores. Sequence-based analysis suggests that one mole of sodium ion is transported along with one mole of cortisol (Na+-dependent cortisol symporter). Therefore, addition of ionophores that bind H+, K+, Ca2+, or Na+ and bypass the transporter through passive diffusion through the plasma membrane should distinguish the ion, if any, that is co-transported with cortisol. Statistical significance between desD + E. coli and desD - control E. coli cells with respect to accumulation of radio-labeled cortisol will be determined by Student t-test. Similar methods will be used to determine effect of ionophore on cortisol transport.The third objective will utilize culture-dependent approaches to isolate and characterize cortisol-metabolizing bacteria in the feces of agriculturally important animals. Feces will be collected and stored at -20°C. One gram of stool will be decimally diluted in anaerobic medium such as peptone-yeast (PY) broth that supports the growth of proteolytic members of the microbiome, particularly Clostridium and Eubacterium spp. some of which have cortisol-metabolizing activity. Higher dilutions that test positive for cortisol-activity assays will be plated on PY agar and other enrichment medias in an anaerobic chamber. Cortisol-metabolizing assay is performed by inoculating each dilution tube into a 1 ml anaerobic vial containing [24-14C] cortisol and incubating overnight at 37°C. [24-14C] cortisol and its metabolites are extracted from the medium with organic solvent, concentrated, and separated by TLC and/or HPLC and compared with migration of known standards of reaction and product purchased from a chemical supplier. Colonies on agar plates will be picked and transferred to broth, once reaching logarithmic phase will be subjected to the cortisol activity assay to identify cortisol-metabolizing colonies from the mixed fecal culture. Colonies exhibiting cortisol metabolism will then be identified to the species/strain level by a combination of biochemical test strips and 16s ribosomal gene sequencing and phylogenetic analysis. The PI has developed gene-specific probes targeting the desA gene that will be used to isolate the corresponding gene in cortisol-metabolizing isolates. Further probe-design and polymerase chain reaction/sequencing will provide the remainder of the sequence of the desAB genes. Sequences from isolates taken from the same host will be compared by computational algorithms (Clustal Omega) and between hosts in order to locate regions in the gene from which oligonucleotides can be designed to quantify these bacteria in fecal samples.

Progress 11/12/15 to 09/30/20

Outputs
Target Audience:Members of the target audience included animal scientists, microbiologists, and molecular biologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and two postdoctoral fellows have trained in anaerobic bacteriology, DNA isolation, polymerase chain reaction, CRISPR-Cas 9 genome engineering, recombinant protein expression and purification, enzymology, and transcriptomics. How have the results been disseminated to communities of interest?We have published manuscripts in high impact peer-reviewed journals, and given invited talks to universities and conferences. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We discovered that bacteria steroid-17,20-desmolase is capable of side-chain cleavage of pharmaceutical cortiocosteroids such as prednisone and dexamethasone (MajorGoal1), and that these metabolites cause proliferation of prostate cancer cells, presumably by signaling through the androgen-receptor. We have also sequence the genome of a novel isolate of Clostridium scindens from pig GI tract and performed transcriptomic analysis in the presence of steroids (Major Goal 4). Starting in March, we began characterization of the cortisol transporter, DesD, by fusing with N-terminal signal peptide, and measuring expression through C-terminal fusion to green fluorescent protein (GFP) (Major Goal 3). This work is ongoing. We are also engineering the desABCD genes into E. coli for upcoming gnotobiotic piglet research.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Ridlon JM, Devendran S, Alves JM, Doden H, Wolf PG, Pereira GV, Ly L, Volland A, Takei H, Nittono H, Murai T, Kurosawa T, Chlipala GE, Green SJ, Hernandez AG, Fields CJ, Wright CL, Kakiyama G, Cann I, Kashyap P, McCracken V and Gaskins HR. 2020. The 'in vivo lifestyle' of bile acid 7 alpha-dehydroxylating bacteria: Comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice. Gut Microbes. 2020 May 3;11(3):381-404. doi: 10.1080/19490976.2019.1618173.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Ly LK, Rowles JL 3rd, Paul HM, Alves JMP, Yemm C, Wolf PM, Devendran S, Hudson ME, Morris DJ, Erdman JW Jr. and Ridlon JM. 2020. 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. J Steroid Biochem Mol Biol. 2020 May;199:105567. doi: 10.1016/j.jsbmb.2019.105567. Epub 2019 Dec 20. PMID: 31870912; PMCID: PMC7333170.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Streidl T, Karkossa I, Segura Mu�oz RR, Eberl C, Zaufel A, Plagge J, Schmaltz R, Schubert K, Basic M, Schneider KM, Afify M, Trautwein C, Tolba R, Stecher B, Doden HL, Ridlon JM, Ecker J, Moustafa T, von Bergen M, Ramer-Tait AE and Clavel T. 2020. The gut bacterium Extibacter muris produces secondary bile acids and influences liver physiology in gnotobiotic mice. Gut Microbes. 2020;13(1):1-21. doi: 10.1080/19490976.2020.1854008. PMID: 33382950.
• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Wolf PG, Devendran S, Doden HL, Ly LK, Moore T, Takei H, Nittono H, Murai T, Kurosawa T, Chlipala GE, Green SJ, Kakiyama G, Kashyap P, McCracken VJ, Gaskins HR, Gillevet PM and Ridlon JM. 2020. Berberine alters gut microbial function through modulation of bile acids. BMC Microbiol. 2021 Jan 11;21(1):24. doi: 10.1186/s12866-020-02020-1. PMID: 33430766.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Members of the target audience included microbiologists and molecular biologists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Several undergraduate students, graduate students, and postdoctoral fellows were trained and presented their work atscientific conferences. How have the results been disseminated to communities of interest?In the form of peer-reviewed high impact publications. What do you plan to do during the next reporting period to accomplish the goals?I have submitted an NIH grant to continue gene discovery. I was awarded an NIH RO1 to study the cortisol degradationpathway in germ-free pigs. This work starts this month (February of 2020).

Impacts
What was accomplished under these goals? We have discovered and characterized numerous steroid metabolizing genes and determined the effect of some of the endproducts on the host that are described in the listed publications.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Devendran S, Shrestha R, Alves JMP, Wolf PG, Ly L, Hernandez AG, M�ndez-Garc�a C, Inboden A, Wiley J, Paul O, Allen A, Springer E, Wright CL, Fields CJ, Daniel SL, Ridlon JM. 2019. Appl Environ Microbiol. scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids. 2019 Mar 22;85(7).
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Doden HL, Pollet RM, Mythen SM, Wawrzak Z, Devendran S, Cann I, Koropatkin NM, Ridlon JM. 2019. Structural and biochemical characterization of 20beta-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32. J. Biol. Chem. 2019 Aug 9;294(32):12040-12053.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Ridlon JM, Devendran S, Alves JM, Doden H, Wolf PG, Pereira GV, Ly L, Volland A, Takei H, Nittono H, Murai T, Kurosawa T, Chlipala GE, Green SJ, Hernandez AG, Fields CJ, Wright CL, Kakiyama G, Cann I, Kashyap P, McCracken V, Gaskins HR. 2019. The 'in vivo lifestyle' of bile acid 7alpha-dehydroxylating bacteria: Comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice. Gut Microbes. 2019 Jun 9:1-24.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Ly LK, Rowles JL 3rd, Paul HM, Alves JMP, Yemm C, Wolf PM, Devendran S, Hudson ME, Morris DJ, Erdman JW Jr, Ridlon JM. 2019. 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. J. Steroid Biochem. Mol. Biol. 2019 Dec 20;199:105567.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience reached by the efforts during this reporting period were scientists and undergraduate students. Some of the results obtained as a result of the studies supported were used as examples in the classroom to teach students at UIUC biochemistry. The results of the studies during the reporting period were published in peer reviewed journals, and also presented at conferences in the United States, Scotland, and Germany. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A masters degree student, Sean Mythen, recently graduated (May 2018) with five co-authored manuscripts published. This Hatch grant has allowed training of a postdoctoral fellow, another masters degree student, and two doctoral students. In addition, we have had four undergraduate students training in the lab. How have the results been disseminated to communities of interest?We have published 5 manuscripts relating to the project during the funding period. In addition, I have presented work relating to the project at the University of Florida, the INRA-Rowett Conference on Gastrointestinal Microbiology in Scotland, and Seeon Conference on Gut Microbiology, Munich, Germany. What do you plan to do during the next reporting period to accomplish the goals?The goal during the next period will be to isolate diverse microbes from livestock that metabolize steroids. We have several manuscripts ready for publication that are expected to be published during the next reporting period.

Impacts
What was accomplished under these goals? Major Goal One has been largely completed. We published extensive enzymatic characterization of the steroid-17,20-desmolase (DesAB). We have also determined structure/mechanism of the accessory enzymes DesC and DesE, which enzyme 20alpha-hydroxysteroid dehydrogenase and 20beta-hydroxysteroid dehydrogenase, respectively. This will be submitted for publication in 2019.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Devendran, S., Mythen, S.M. and Ridlon, J.M. 2018. The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase. J Lipid Res. 2018 Jun;59(6):1005-1014.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Doden, H., Sallam, L.A., Devendran, S., Ly, L., Doden, G., Daniel, S.L., Alves, J.M.R. and Ridlon, J.M. Metabolism of oxo-bile acids and characterization of recombinant 12alpha-hydroxysteroid dehydrogenases from bile acid 7alpha-dehydroxylating human gut bacteria. Appl Environ Microbiol. 2018 May 1;84(10). pii: e00235-18.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Harris, S.C., Devendran, S., Alves, J.M.P., Mythen, S.M., Hylemon, P.B. and Ridlon, J.M. 2018. Identification of a gene encoding a flavoprotein involved in bile acid metabolism by the human gut bacterium Clostridium scindens ATCC 35704. Biochim Biophys Acta 2018;1863(3):276-283. PMID: 29217478.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Morris, D.J. and Ridlon, J.M. 2017. Glucocorticoids and gut bacteria: The GALF Hypothesis in the metagenomic era. Steroids 2017; 125:1-13. PMID: 28624548.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Harris, S.C., Devendran, S., M�ndez-Garcia, C., Mythen, S.M., Wright, C.L., Fields, C.J., Hernandez, A.G., Cann, I., Hylemon, P.B. and Ridlon, J.M. 2018. Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243T. Gut Microbes. 2018 Apr 4:1-17.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Mythen, S.M., Devendran, S., M�ndez-Garcia, C., Cann, I. and Ridlon, J.M. 2018. Targeted synthesis and characterization of a gene-cluster encoding NAD(P)H-dependent 3alpha-, 3beta-, and 12alpha-hydroxysteroid dehydrogenases from Eggerthella CAG:298, a gut metagenomic sequence. Appl Environ Microbiol. 2018 Mar 19;84(7). pii: e02475-17. PMID: 29330189.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Members of the target audience included microbiologists, biochemists, physicians, and animal scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?My graduate student learned kinetic analysis, site-directed mutagenesis, isothermal titration calorimetry, crystallization robotics, and X-ray diffraction (at Nicole Koropatkin's lab at the University of Michigan). My postdoctoral fellow trained my student and is now in a good position to publish several high impact papers. How have the results been disseminated to communities of interest?Through peer-reviewed publications as well as invited talks (Baylor University School of Medicine, University of Toledo, Beckman Institute of Advanced Study, UIUC, Mayo Clinic, National Institutes of Health, etc). What do you plan to do during the next reporting period to accomplish the goals?We will begin working on a genetic system and sequence genomes of organisms with this pathway so that we can develop mechanistic animal models to determine the importance of this pathway.

Impacts
What was accomplished under these goals? Major Goal 1 is nearly complete. The three proteins that convert cortisol to 11β-hydroxyandrostenedione include DesC, DesE, and DesAB. We are currently writing a manuscript on the 2.0 Å structure of DesC along with advanced quantum mechanical molecular modeling (QM/MM) allowing us to dock cortisol into the active-site, and the mechanism and free-energy of the transition state and the overall reaction. We tested this mechanism and confirmed it through a combination of site-directed mutagenesis, isothermal titration calorimetry, and circular dichroism spectroscopy. We expect this to be published in a Nature journal or ACS Catalysis in 2018. We have solved the crystal structure of DesE to 2.1Å and have fully characterized this enzyme kinetically along with site-directed mutagenesis and will perform additional ITC experiments and work to co-crystalize cofactor and substrate. We have also confirmed that the DesAB is the steroid-17,20-desmolase, and we developed a coupled assay to directly measure the activity spectrophotometrically by addition of a recombinant 17beta-hydroxysteroid dehydrogenase which utilizes NADPH to reduce the 17 ketone of the resultant product of the DesAB conversion of cortisol. We are preparing to write a manuscript to be published in the Journal of Lipid Research.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Harris, S.C., Devendran, S., Alves, J.M.P., Mythen, S.M., Hylemon, P.B. and Ridlon, J.M. 2018. Identification of a gene encoding a flavoprotein involved in bile acid metabolism by the human gut bacterium Clostridium scindens ATCC 35704. Biochim. Biophys. Acta. 2017 Dec 4. pii: S1388-1981(17)30234-2.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Morris, D.J. and Ridlon, J.M. 2017. Glucocorticoids and gut bacteria: The "GALF Hypothesis" in the metagenomic era. Steroids. 2017 Sep;125:1-13.
• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Sean Mythen, Saravanan Devendran, Celia M�ndez-Garc�a, Isaac Cann and Jason Ridlon. 2018. Targeted synthesis and characterization of a gene-cluster encoding NAD(P)H-dependent 3alpha-, 3alpha-, and 12alpha-hydroxysteroid dehydrogenases from Eggerthella CAG:298, a gut metagenomic sequence.

Progress 11/12/15 to 09/30/16

Outputs
Target Audience:The target audiences for the current reporting period included scientists and clinicians (data from this project was presented at a Harvard Probiotics Meeting, as well as students in formal classroom instruction. Data obtained as part of this Hatch project fit very nicely into the biochemistry course (Ansci 350) that I taught during Fall 2016. This includes protein structural data, molecular docking experiments, protein purification and electrophoresis, as well as enzyme kinetic analysis. During the of Spring 2017 I will be co-teaching a gastrointestinal microbiology course in which I will be incorporating lecture material on steroid metabolism in the gut, and will utilize data obtained from these studies. Changes/Problems:Unexpected outcomes: In searching for steroid-17,20-desmolase genes, we identified a normal inhabitant of the urinary tract encoding the desABE genes. We obtained this strain and confirmed steroid-17,20-desmolase activity. Production of androgens in the urinary tract by bacteria has not been reported, and may have implications for reproductive health. Signficant Deviations: Our focus this reporting period has not been on steroid-17,20-desmolase itself, which is the focus of Aim 1. Instead, we have been focusing on the 20-alpha and 20-beta-hydroxysteroid dehydrogenases (HSDH). We found that Bifidobacterium spp. inhabiting the gut encode 20beta-HSDH and we suspect that this might have probiotic function in prevention of androgen and progestin formation by other gut bacteria. During the next reporting period we will be characterizing steroid-17,20-desmolase. We have submitted a manuscript on the 20beta-HSDH and will be submitting another manuscript during the next reporting period on 20alpha-HSDH structure and catalytic mechanism. Delay: The putative cortisol transporter gene (desD) was cloned into an expression vector and attempts have been made to overexpress in E. coli. Using the particular vector, strain of E. coli, and conditions we were unable to observe expression of the transporter. Since we were making good progress with enzymes in this pathway, we have decided during this reporting period to postpone work on the transporter until the other work has been published. We will resume work on DesD in the near future. What opportunities for training and professional development has the project provided?This project allowed training opportunities for undergraduate research students, a graduate student and a postdoctoral fellow. My postdoctoral fellow has trained in an RNA Sequencing workshop put on by the Keck Center for Biotechnology on campus. Both myself and my postdoctoral fellow participated in a week-long workshop put on by the Theoretical and Computational Biophysics Group at the Beckman Institute for Advanced Study at UIUC. The workshop focuses on programs allowing visual representation, modeling, and simulation of macromolecules. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, my laboratory will work on characterization of the desAB gene products, encoding the steroid-17,20-desmolase. We will also continue work on the DesD, the putative cortisol transport protein. We will also begin working on the isolation of organisms capable of steroid-17,20-desmolase from production animals.

Impacts
What was accomplished under these goals? We have made substantial progress on Problem statement 1 including successful overexpression and purification of the DesA and DesB gene products in E. coli. We have solved the crystal structure of the DesC gene and performed molecular docking experiments fitting cortisol and cofactor NAD in the active-site, a collaborative effort with the Theoretical and Computational Biophysics Group at UIUC. We have successfully performed site-directed mutagenesis on putative active-site residues as well as zinc-finger motifs important for quaternary structure. These studies are important for identification of defining residues in 20-HSDH enzymes in sequencing data from gut isolates and metagenomic sequences of gastrointestinal samples. We have also identified steroid-17,20-desmolase genes in Butyricicoccus desmolans and Clostridium cadavaris and characterized a novel 20beta-hydroxysteroid dehydrogenase (HSDH) (DesE) associated with DesA and DesB in these organisms. Our phylogenetic analysis revealed members of the bifidobacteria that encode 20beta-HSDH but not DesA and DesB. Unexpectedly, this analysis also identified DesABE in a member of the urinary microbiota, Propionimicrobium lymphophilum. The desD gene has been cloned into pSport-1 and attempts have been made to overexpress this protein; however, additional work must be done to determinet the conditions (i.e. host strain, vector, growth medium, temperature, etc) that will allow expression of this membrane transport protein.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2017 Citation: Devendran, S., Mendez-Garcia, C. and Ridlon, J.M. 2017. Characterization of a recombinant 20beta-hydroxysteroid dehydrogenase from the anaerobic gut bacterium Butyricicoccus desmolans ATCC 43058. Submitted to Applied Environmental Microbiology.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Ridlon, J.M., Devendarn, S., Bernardi, R.C., Bhowmik, S., Cann, I.K.O., Schulten, K.J., Lesley, S.A. and Mythen, S.M. 2016. Characterization of bacterial 20-hydroxysteroid dehydrogenases: Potential probiotic function in essential hypertension? Gut Health, Microbiota & Probiotics Throughout the Lifespan: Metabolic & Brain Function, 2016, Harvard Medical School, Boston MA, USA.
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Morris, D.J. and Ridlon, J.M. 2017. Glucocorticoids and gut bacteria: Is essential hypertension a bacterial disease? Submitted to Steroids.