Cormac Gahan APC, University College Cork, Ireland

Transcription

1 Valencia, 2015 Bile Research Group Bacterial Bile Salt Hydrolase in the Regulation of Host Lipid Metabolism & Circadian Rhythm: A Role in Probiotic Function? Cormac Gahan APC, University College Cork, Ireland c.gahan@ucc.ie Dr. Susan Joyce

2 Microbe:Host Interactions Bile Acids Vinolo et al. Nutrients 2011, 3(10),

3 Bile in Systemic Microbe-Host Signalling & Bile acids act here Bile acids act here

4 Endocrine function: energy metabolism Bile Bile Bile Bile Bile Bile

5 BSH function: a probiotic effector? Probiotics: Potential to change this signalling landscape?

6 Bacterial bile acid modifications in the gut: Taurine or Glycine

7 Complexity: Cholesterol CA Primary BAs CDCA a-mur b-mur DCA LCA 7-keto LCA W-Mur T-DCA T-CA Secondary BAs T-LCA T-CDCA T-a-Mur T-b-Mur G-DCA G-CA G-LCA G-CDCA T-wMCA S-LCA UDCA Tertiary BAs

8 Bile Acids: Wider potential significance 2. Reabsorbed bile salts as signalling molecules regulating endocrine functions (obesity) Watanabe et al Nature, 439: Bile acids as local signalling molecules regulating innate immunity (Angiogenin synthesis and inos in the ileum) - FXR Inagaki et al. (2006) PNAS, 103:

9 PstI MCS Cloning of BSH in E. coli MG1655: glms Tn7L Tn7R WGM R 30 nt 122 nt yiea MG1655Str R ::GM2 BSH1 allele JCM1046 BSH2 allele UCC118 AflII AflII P 44 P 44 ATG ATG BSH1 BSH2 TAA TAA SacII SacII MG1655Str R ::GM2BSH1 MG1655Str R ::GM2BSH2 Streptreated mice Miranda et al Infect Immun 72: MG1655 (red) Eubacteria (green)

10 In vitro human bile assays BSH in E. coli is working E. coli 1% Bile _017 1: TOF MS ES TIC Conjugated e % 0 ex vivo bile acid incubation Time E. coli +BSH1 Treated Unconjugated MB62_ _007 1: TOF MS ES TIC e6 % Time

11 E. coli strain colonises the mouse gut Cronin M. et al PLoS One. 2012;7(1):e30940.

12 EC-BSH1 significant in vivo activity o o Taurine Bile salt hydrolase Tauro-conjugated Bile Total Plasma Tauro-Bile Acids Unconjugated Bile Reduction in TbMCA

13 EC-BSH1 reduced weight gain Weight Gain Cholesterol EC ECBSH1 No bacteria * P= * P=0.0262

14 Mono-colonize Gnotobiotic Mice with BSH+ or BSH- Mono-colonise Germ-free mice Germ-free ECBSH1 ECBSH2 Conv-D EC all n=5

15 Joyce et al PNAS. 111(20): Host Gene Expression influenced by Bile

16 Role in Reduced Cholesterol & Weight Targets in Conventional Mice BSH: abcg5 RCT and TICE TICE Trans-intestinal cholesterol excretion (TICE) Reverse Cholesterol transport (RCT) Reduces lipid biosynthesis RCT Hepatobiliary excretion

17 DCA and CDCA induce lipid signalling genes in Caco2 cells RT-PCR 18 hours Gut bacteria CDCA BSH TCDCA RCT and TICE Apolipoproteins Enzymes Currently examining functional lipid transport using a transwell system ABC Transporters

18 Circadian Genes Switch by BSH1 in mice EC BSH1 EC BSH1

19 Relative mrna expression CACO-2 Circadian Genes Influenced in vitro Gut bacteria 14 Per2 CDCA BSH TCDCA Caco2 DMSO DCA CDCA TDCA TCDCA Time in hours Unconjugated bile acids enhance periodic expression of circadian genes in vitro Dr. Kalai Govindarajan

20 Oral gavage alters ileal circadian gene expression Unconjugated bile acids enhance periodic expression of circadian genes in vivo Dr. Kalai Govindarajan

21 Hypothesis: BSH activity alters peripheral circadian clock Well known that bile synthesis is governed by circadian rhythm We show that bacterial activity alters the signalling potential of bile with an influence upon peripheral rhythms CDCA DCA Gut bacteria BSH TDCA TCDCA Potential for probiotic intervention? Thaiss CA et al Cell. Diurnal oscillations in gut bacteria!

22 BSH role in local homeostasis? Conventionally-raised mice in mice EC BSH1 BSH2 ConvD

23 Conclusions BSH important for bacterial colonisation (mutualism?) Expression of BSH (single gene/function) in the gut significantly influences local and systemic responses Allelic variation was significant (BSH1 versus BSH2) Expression of BSH1 influences body weight and serum cholesterol - possible pathways identified Joyce et al PNAS. 111(20):

24 Bile conversions: Multiple Effects Upon the Host Linking microbiota, bile, weight gain: Joyce et al PNAS. 111(20):

25 Functional Annotation of the Microbiome Systems Approach: What s there? Reductionist: What are they doing? Culture-independent (sequencing, proteomics) Single protein effects on system Single organism / protein

26 Future? Selecting Probiotics UPLC-MS analysis of bile conversions Human Bile v v v v E.coli (EC) ECpNZ44 ECpBSHa ECpBSHb ECpBSHc ECpBSHd ECpmBSH1 ECpmBSHe ECpNZ44 ECpmBSHf ECpmBSHg ECpmBSHh Profiling cloned BSHs and whole probiotics (left) Some probiotics have BSH but no demonstrable activity Sarah Louise Long

27 Correlate probiotic BSH activity with effects on adiposity? Probiotics selected on the basis of BSH activity show a trend towards inducing weight loss in mice Currently testing in a diet-induced obesity model

28 On-going work: Mechanisms/pathways/circadian rhythm Relating BSH structure to function & selection of probiotics Analysis of bile acid metabolism in human disease states

29 Gut Microbes Addendum bacterial bile salt hydrolase in host metabolism.. Original Photo: Dr. Debbie Watson

30 Acknowledgements Dr. Susan Joyce Dr. John MacSharry Dr. Kalai Govindarajan Pat Casey Dr. Michael Kinsella Dr. Eileen Murphy Sarah-Louise Long Dr. Susan Joyce Prof. Colin Hill Prof. Fergus Shanahan Dr. Julian Marchesi Dr. Brian Jones Dr. Paul Cotter Frances O Brien & Staff of BSU Dr. Carthage Moran Dr. Marcus Claesson

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32 Take home message & Bile acids act here Intervention? Bile acids act here

33 et al. 2015

34 UPLC-MS UPLC-MS Approach to detect 30 Bile Acids

35 Intervention Alters Plasma Bile Acid Profiles in Mono-colonised Mice: Total Tauro-Conjugated Plasma Bile Acids Reduction in TbMCA

36 Intestinal Bacteria microbiota a hidden organ More bacteria (10 14 ) than human cells (10 13 ) in the body Approx 2000 bacterial phylotypes in human intestine Significant metabolic activity

37 The Human Superorganism Microbiologists Our Time Has Come! Bacteria Metabolism > Liver Energy extraction fat deposition Immune stimulation Barrier to infection

38 Early study Eckburg PB. et al Diversity of the human intestinal microbial flora. Science 308:

39 Log 10 CFU/ml BSH enhances microbial bile resistance Cloning and expression of gut BSH enzymes in Listeria innocua: BSH enhances survival oflisteria innocua in bovine bile: ve BSH type A +ve BSH type D * * * * * BSH type E BSH type F ve BSH D BSH F BSH A BSH E + ve Clone

40 BSH enhances gut colonization in conventional mice (day 3 PI) BSH expressed in Listeria innocua: CFU/ Intestine 10 9 N=5, * p< * * * * Clone Jones, B, Begley, M et al, Proc Natl Acad Sci USA, 105:

41 Microbial Bile salt hydrolase activity o Cormac Gahan Hypothesis: R glycine BSH is uniquely o associated with the Bile gut salt microbiota & influences host physiology hydrolase Conjugated Bile acid Susan Joyce mice taurine or Unconjugated Bile acid Models to investigate BSH activity in BSH catalyses the gateway reaction Bsh + Bsh Bsh - - dehydroxylation dehydrogenation Precipitate on Tauro-Bile plates

42 Functional Metagenomics - diversity of BSH activity in gut bacteria Dr. Brian Jones & Dr. Julian Marchesi Metagenomic clones expressing BSH activity High Mw DNA was extracted from a faecal sample from a healthy male Fosmid bank consisting of metagenomic clones

43 Functional Annotation of the Microbiome Systems Approach: What s there? Reductionist: What are they doing? Culture-independent (sequencing, proteomics) Single protein effects on system Single organism / protein

44 Intervention Alters Plasma Bile Acid Profiles in Mono-colonised Mice: Total Tauro-Conjugated Plasma Bile Acids Reduction in TbMCA

45 Phylogenetic analysis of 400 PVA-BSH family sequences host-level selection in the gut A 2 B 1 A 1 Tauro-CBA activity 1 2 B Glyco- and Tauro-CBA activity Gut metagenomes Sargasso sea metagenome 3 C 1 C 2 C 3 No activity against bile BSH - associated only with bacteria from the gut or gut pathogens (groups A and B) A related enzyme - PVA is present in gut & non-gut bacteria but does not have BSH activity (group C) Gut Archea (marked 1 and 2) have evolved potent BSH activity whereas non-gut Archea (marked 3) do not express active BSH Proc Natl Acad Sci U S A (36):

46 E. coli MG1655strep R colonises the mouse GI tract 60 days - Strep Cronin M. et al PLoS One. 2012;7(1):e30940.

47 Quantitative UPLC-MS Internal Standards: Deuterated Chenodeoxycholic acid and Cholic acid 20 ug/ml + IS _008 T-B-MCA CA LCA % Taurine 0.75 T-DCA A-MCA T-CA UDCA B-MCA 8.46 CDCA T-LCA DCA