Metabolite cross-feeding among the human gut microbiota

Transcription

1 Metabolite cross-feeding among the human gut microbiota Harry J Flint Sylvia H Duncan Karen P Scott Petra Louis Microbiology Group, Rowett Institute, University of Aberdeen,UK

2 Metabolite cross-feeding in anaerobic gut communities the rumen Fermentation products Hydrogen (and formate) transfer Lactate, succinate, acetate Products of substrate degradation Polysaccharides, oligosaccharides, sugars, phenolic compounds Vitamins, cofactors, biosynthetic precursors eg. vitamins, branched chain fatty acids, haem (Flint HJ 1997 TIM 5, )

3 Interspecies hydrogen transfer - Mike Wolin, Marvin Bryant 1. 2 ethanol CO 2 2 XH 2 2 acetaldehyde 2 H 2 H 2 O 2 acetate 2 H 2 2 XH 2 S organism H 2 O methanogen CH 4 2. glucose CO 2 2 NADH 2 2 ATP 2 pyruvate 2 H 2 H 2 O Ruminococcus albus 2 CO 2 2 FdH 2 2 acetyl-coa 2 H 2 H 2 O methanogen 2 ATP 2 acetate CH 4

4 Syntrophy dependent on H (or formate) consumption - S organism first example of an obligate syntroph, ie. growth thermodynamically impossible without the second organism. 2 ethanol to 2 acetate:- G +19 kj (no methanogen) G -112 kj (+ methanogen) [First isolated as a coculture - Methanobacillus omelianski in 1939] Some other examples of syntrophy (diverse habitats)*:- substrates used in co-culture...in pure culture Obligate syntrophs Syntrophomonas sapovorans Fatty acids (C4-C18, C16:1, C18:1, C18:2) None? Pelotomaculum schinkii Propionate (C3) None? Non-obligate syntrophs Syntrophobacter wolinii Propionate (C3) C3 + sulfate; fumarate Syntrophomonas bryantii Fatty acids (C4-C11) crotonate (C4:1) Sporotomaculum syntrophicum Benzoate crotonate; crotonate + benzoate *[McInerney et al (2008) Ann NY Acad Sci 1125: 58-72]

5 Fermentative metabolism of Ruminococcus albus (Wolin & Miller, 1983) (A) 1 glucose (B) 1 glucose 2 ADP 2 NAD + 2 ADP 2 NAD + 2 Fd red 2-2 ATP 2 NADH 2 ATP 2 NADH 2 Fd ox 2 pyruvate 2 H 2 2 pyruvate 4 H 2 2 CoA 2 Fd ox 2 CoA 2 Fd ox 2 CO 2 2 Fd red 2-2 CO 2 2 Fd red 2-1 ethanol 2 acetyl-coa 2 acetyl-coa 2 NAD + 2 NADH 1 Pi 1 CoA 2 Pi 2 CoA 1 acetyl-p 2 acetyl-p 1 ADP 1 ATP 2 ADP 2 ATP 1 acetate 2 acetate No hydrogen consumption hydrogen consumption Slide - courtesy of Petra Louis

6 Acetogenesis [1 glucose to 3 acetate] 1 glucose 2 ADP 2 NAD + 2 ATP 2 pyruvate 2 NADH 2 CoA 2 Pi 2 CoA 2 acetyl CoA 2 acetyl-p 4 [H] 2 [H] 2 [H] CO [H] + CO [H] Wood-Ljungdahlpathway - abundant in human colonic metagenome [Rey FE et al (2009) J Biol Chem] 2 ADP 2 ATP 2 acetate 1 acetate eg. Blautia hydrogenotrophica (formerly Ruminococcus hydrogenotrophicus) [Bernalier A et al (1996) Arch Microbiol]

7 Hydrogen utilizers in the human colon Methanogens (eg. Methanobrevibacter smithii Archaea) Approx. 1 in 3 of people are methanogenic 1 (breath H 2 ): CH 4 producers harbour >10 8 methanogens/ ml faeces 2 CH 4 non-producers harbour / ml faeces 2 Acetogens (eg. Blautia hydrogenotrophica Lachnospiraceae) Acetogenesis can contribute 30% of acetate C ( 13 CO 2 ) 3. Sulphate-reducers ( eg, Desulphovibrio piger - Proteobacteria) Possible association with colitis? These three groups compete for hydrogen, but may also co-exist 2,4,5,6 [ 1 Levitt et al., 2000, Am J. Gastr; 2 Dore et al., 1995, FEMS ME; 3 Miller & Wolin 1996, AEM; 4 Bernalier et al., 1996, FEMS ME; 5 Gibson et al., 1993, FEMS ME; 6 Nava et al., ISME J]

8 Cross-feeding in short chain fatty acid metabolism 1. Carbohydrate fermentors widespread - Bacteroidetes, Firmicutes, Actinobacteria Carbohydrates (hexoses) 3. Acetogens Blautia hydrogenotrophica Marvinbryantia formatixigenes (Firmicutes) 4. Methanogens Methanobrevibacter smithii (Archaea) 1 pyruvate acetyl CoA 5. Sulfate reducers Desulfovibrio piger (Proteobacteria) 2. Propionate producers Bacteroidetes Veillonellaceae CH 4 4 succinate lactate formate/ H 2 + CO 2 3 ACETATE 2 butyryl CoA 6 5 PROPIONATE SO 4 H 2 S BUTYRATE 6. Butyrate producers Faecalibacterium prausnitzii Eubacterium rectale, Roseburia spp. Eubacterium hallii, Anaerostipes spp. (Firmicutes)

9 Dominant bacterial species Walker AW et al ISME J (2011) - 26 faecal samples from 6 obese males 295 other phylotypes (72% uncultured) 25 cultured species accounted for approximately 50% of 16S rrna sequences :- (from - Flint HJ et al Gut Microbes, 2012) Faecalibacterium prausnitzii Eubacterium rectale Colinsella aerofaciens Clostridium clostridioforme Bacteroides vulgatus Anaerostipes hadrus Ruminococcus bromii Eubacterium hallii Blautia wexleri Bacteroides dorei Roseburia faecis Dorea longicatena Subdoligranulum variabile Bacteroides uniformis Ruminococcus obeum Bacteroides ovatus Blautia luti Parabacteroides distasonis sp nov A2-166 sp nov SR1/5 Lachnospira pectinoschiza sp nov 80/3 Dialister invisus Roseburia inulinivorans Ruminococcus callidus others

10 Dominant bacterial species butyrate-producers Walker AW et al ISME J (2011) - 26 faecal samples from 6 obese males Faecalibacterium prausnitzii Eubacterium rectale Colinsella aerofaciens Clostridium clostridioforme Bacteroides vulgatus Anaerostipes hadrus Ruminococcus bromii Eubacterium hallii Blautia wexleri Bacteroides dorei Roseburia faecis Dorea longicatena Subdoligranulum variabile Bacteroides uniformis Ruminococcus obeum Bacteroides ovatus Blautia luti Parabacteroides distasonis sp nov A2-166 sp nov SR1/5 Lachnospira pectinoschiza sp nov 80/3 Dialister invisus Roseburia inulinivorans Ruminococcus callidus others

11 Butyrate metabolism Carbohydrates, amino acids pyruvate lactate Eubacterium hallii Anaerostipes spp acetyl-coa acetate butyryl CoA:acetate CoA transferase Faecalibacterium prausnitzii Eubacterium rectale, Roseburia spp. Eubacterium hallii, Anaerostipes spp. butyryl-coa butyrate butyrate kinase Coprococcus spp. [Louis P et al J Bacteriol 4004 Duncan SH et al AEM 2004]

12 Butyrate formation in Roseburia spp., Eubacterium rectale and Faecalibacterium prausnitzii 8 ADP 8 ATP 4 glucose 8 NAD + 8 NADH 8 pyruvate 8 CoA 8 Fd ox 3 ADP 3 ATP 5 butyrate 2 acetate 5 acetate 5 Fd ox 10 NADH 5 Etf 10 NAD + 8 CO 2 3 CoA 3 Pi 3 acetyl-p 8 acetyl-coa 5 acetyl-coa 5 butyryl-coa BCD THL 5 acetoacetyl-coa 5 EtfH 2 5 crotonyl-coa 5 H 2 O 5 NADH 5 NAD hydroxybutyryl-CoA CRO 5 CoA BHBD 6 H 2 6 Fd ox 13 Fd red 2-7 NAD + 7 NADH 7 Fd ox cytoplasm membrane 14 H + (Louis P & Flint HJ FEMS ML 2009)

13 Co-culture with an acetogen xylan Roseburia intestinalis (lactate) H 2 + CO 2 (+ formate) Blautia hydrogenotrophica mm Chassard et al., 2006 FEMS ML acetate butyrate H2 CH4 butyrate acetate Co-culture with acetogen Co-culture with methanogen H 2 acetate butyrate H 2 CH 4 Consequences for energy harvest in the complete ecosystem? [nb. methanogens can coexist with acetogens, but may also slow gut transit]

14 Butyrate formation from lactate and acetate by Eubacterium hallii and Anaerostipes spp. [Duncan et al 2004 AEM 70, ] 4 lactate 8 [H] 2 H 2 4 pyruvate 2 Fd 2 NADH 2 2 NAD 4 Fd 2 FdH 2 2 FdH 2 4 acetyl-coa 3 CoA 6 [H] + 2 acetyl-coa 4 CoA 4 CO 2 Increase in butyrate (mm) Data for 7 lactate-utilizing strains :- ADP ATP acetyl- P 3 H 2 O 6 [H] 3 butyryl-coa acetate 2 acetate Decrease in (Ace + Lac)/2 (mm) CoA acetyl-coa P i 3 butyrate

15 Butyrate formation from lactate and acetate by Eubacterium hallii and Anaerostipes spp. [Duncan et al 2004 AEM 70, ] 4 lactate E. hallii L2-7 A. hadrus SS2/1 2 H 2 4 Fd 8 [H] 4 pyruvate 4 CoA 2 Fd 2 NAD 2 FdH 2 2 FdH 2 4 acetyl-coa 3 CoA + 2 acetyl-coa 4 CO 2 2 NADH 2 6 [H] ATP ADP acetyl- P 3 H 2 O 6 [H] 3 butyryl-coa acetate 2 acetate [Munoz-Tamayo R et al 2011, FEMS Microbiol Ecol] - E. hallii L2-7 uses both D- and L lactate. - A. hadrus SS2/1 uses only D- lactate*. - E. hallii uses less lactate when glucose present. - A. hadrus less affected by glucose. CoA P i acetyl-coa 3 butyrate * D- lactate is a neurotoxin at high concentrations

16 Interactions between lactate-utilizing bacteria Bifidobacterium adolescentis L- lactate + acetate Eubacterium hallii [mm] 20 Bifid alone [Starch supplied as energy source] E.hallii alone Bifid + E.hallii Formate Acetate Butyrate Lactate Sulphide 10 0 butyrate (beneficial) - Bacterial combinations

17 Interactions between lactate-utilizing bacteria Bifidobacterium adolescentis L- lactate + sulphate [mm] 20 Bifid alone [Starch supplied as energy source] E.hallii alone D.piger alone Bifid + E.hallii Bifid + D.piger Formate Acetate Butyrate Lactate Sulphide Bifid + E.hallii + D. piger Desulfovibrio piger 10 0 acetate + H 2 S Bacterial combinations (toxic) (Marquet P et al FEMS ML 2009)

18 ph-induced dysbiosis in vitro - fermentation patterns + polysaccharides [means of 24h incubations with four different faecal inocula] Change in concentration (mm) ph 5.2 ph 5.9 ph 6.4 * * Acetate Propionate Butyrate Lactate * Not detectable Concentration (mm) Lactate accumulation in stool in colitis (Vernia et al 1988) Controls Quiescent colitis Mild colitis Moderate colitis Severe colitis Lactate accumulation shown to be due mainly to reduced lactate utilization by other bacteria at ph 5.2 ( 13 C lactate) [Belenguer et al AEM 2007; also Walker et al AEM 2005, Duncan et al EM 2009]

19 Conclusions Cross-feeding must be assumed to occur widely in any complex microbial community A few syntrophs may not be capable of isolation in pure culture Hydrogen consumption can profoundly affect the energetics and metabolism of hydrogen-producing organisms, and therefore also community composition and metabolic outputs Cross-feeding of acetate, lactate and succinate plays a major role in short chain fatty acid production Cross-feeding of fermentation products can be expected to broaden the impact of probiotics and prebiotics on the gut microbiota

20 Two distinct mechanisms of cross-feeding can be involved in stimulating butyrate formation in cocultures* Starch or FOS Bifidobacterium adolescentis L2-32 Oligosaccharides formate L-lactate acetate fermentation products 2 - substrate spillover butyrate E. hallii Roseburia sp. [* Belenguer A et al., AEM also Falony G et al., AEM 2006)

21 Cross-feeding from the breakdown of complex carbohydrate substrates Insoluble complex carbohydrates Primary degraders Diet Soluble polysaccharides Oligosaccharides, sugars Polysaccharide utilisers H-utilisers (acetogens, methanogens, SRB); lactate utilisers Metabolic products Oligosaccharide/ sugar utilisers (Flint HJ et al Env Micro 2007)

22 Partitioning of bacterial 16S rrna sequences between liquid and particulate fractions of human faecal samples % 16S rrna gene sequences (Means of four samples) P = P = ruminococci on faecal fibre Liquid Particles R-ruminococci are preferentially associated with fibre particles in stool samples. Bacteroidetes partition more into the liquid phase [Walker AW et al Env Microbiol (2008)]

23 Degradation of four types of corn starch by four amylolytic human gut bacteria % starch utilization 100% 80% 60% 40% 20% 0% Resistant starches (RS2 S2,S3; RS3 S4) Ruminococcus bromii (Firmicutes) 100% 80% S1 S2 S3 S4 Autoclaved Bifidobacterium adolescentis (Actinobacteria) Grampositive 60% 40% 20% Eubacterium rectale (Firmicutes) 0% 100% 80% S1 S2 S3 S4 Boiled R.bromii B.adol E.rectale B.theta Bacteroides thetaiotaomicron (Bacteroidetes) Gramnegative 60% 40% 20% 0% S1 S2 S3 S4 Raw [Ze X et al 2012 ISME J]

24 Stimulation of starch utilization in vitro by co-incubation with R. bromii L Boiled RS3 [Ze X et al 2012 ISME J] 1500 Total sugar utilized (t48) Reducing sugar released (t48) µg/ml [on YCFA medium] 0 Rum = Ruminococcus bromii Erec = Eubacterium rectale Bac = Bacteroides thetaiotaomicron Bif = Bifidobacterium adolescentis R. bromii (Rum) is unable to grow on YCFA medium, but its enzymes still degrade RS, mainly to maltose and glucose (reducing sugars). As a result, starch utilization by the other three bacteria is greatly stimulated in co-culture with R. bromii.

25 Growth on starch breakdown products - Ruminococcus bromii L2-63 Bifidobacterium adolescentis L2-32 OD OD Time (h) Time (h) R. bromii pullulan malto- malto- maltose glucose panose isomaltose tetraose triose E. rectale B. thetaiotaomicron B. adolescentis

26 Keystone role of Ruminococcus bromii in the fermentation of resistant starch % RS left Utilization of Type 3 RS by human faecal bacteria in vitro :- 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Hour Vol 2 Vol 6 Vol 7 Vol 25 Bacterial community profiles (16S rrna sequencing) mn. 6 volunteers Firmicutes (-r) R-ruminococci Bacteroidetes Proteobacteria Actinobacteria Effect of adding amylolytic bacteria to faecal incubations in vitro :- (C) 100% v25 100% v7 % of residual starch 80% 60% 40% 20% No addition E. rectale, B. adolescentis or B. thetaiotaomicron + R. bromii % of residual starch 80% 60% 40% 20% 0% 0% Time (h) Time (h) [Ze X et al ISME J 2012; Walker AW et al ISME J 2011]

27 Microbial conversion of plant-derived phenolics community pathway? ferulic acid HO O HO hydrogenation O HO demethylation O HO dehydroxylation O Faecal concentrations (human dietary study) [Russell WR et al AJCN 2011] Concentration (µg cm -3 ) O O HO HO OH OH OH 25 P = M HPMC 20 HPLC P < P < ferulic acid metabolite 1 metabolite 2 metabolite 3

28 Cross-feeding of growth factors & vitamins External supply of redox reactants (eg. flavins)? MT Khan et al The gut anaerobe Faecalibacterium prausnitzii uses an extracellular electron shuttle to grow at oxic-anoxic interphases. ISME J (2012) No bacteria Roseburia inulinivorans Faecalibacterium prausnitzii Oxygen Gradient Colonocytes Oxygenated zone Mucus Transition Zone Fecal cylinder Dependent on thiols (eg. glutathione) + redox reagent (eg. riboflavin) O 2 H 2 O RS-SR 2R-SH red. flavins ox. flavins bacterial cell

29 Networks of association suggested from metagenomic output. But do they indicate - inter-species interactions? - similar responses to gut environment/diet/host? Further conclusions For some recalcitrant substrates (eg. plant cell walls, mucin, resistant starch) specialised keystone species facilitate utilization by the rest of the community Cross-feeding can refer to almost any interaction whereby a product of one organism affects the growth and metabolism of another member of the microbial community The future - is highly interactive! We already need theoretical modelling to predict the outcomes of such interactions in complex communities

30 Acknowledgements RINH Microbial Ecology Group Sylvia Duncan Petra Louis Karen Scott Jenny Martin Freda McIntosh Alan Walker Cedric Charrier Xiaolei Ze Alvaro Belenguer Jennifer Ince Lucy Webster Masa Vodovnik Anna Szcepanska Biomathematics and Statistics Scotland Grietje Holtrop, Helen Kettle Rowett Institute of Nutrition and Health Obesity and Metabolic Health Human Nutrition Unit Chemical Analysis Alexandra Johnstone Sylvia Hay David Brown Gerald Lobley Clair Fyfe World Cancer Research Fund and Scottish Government (RSD) support

31 Collaborators Wellcome Trust Sanger Institute, UK Alan Walker Trevor Lawley Keith Turner Julian Parkhill U. Groningen, Netherlands Hermie Harmsen INRA Theix, France Annick Bernalier Christophe Chassard U. Dundee, UK George Macfarlane Sandra MacFarlane EU SATIN consortium Wageningen, Netherlands Jerry Wells Willem de Vos U. Girona, Spain Mireia Lopez-Siles Jesus Garcia-Gill Helsinki, Finland Anne Salonen U. Illinois, USA Bryan White Weizmann Institute, Israel Ed Bayer Tel Aviv U, Israel Raphael Lamed EU FibeBiotics consortium