http://apc.ucc.ie

Microbial metabolite production by gut microbes and implications

for host health

Catherine StantonAPC Microbiome Institute (APC) & Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork,

Ireland.

E-mail: catherine.stanton@teagasc.ie

http://apc.ucc.ie

Bacterial Community

Microbiota~100 trillion cells~3 million genes

Human~10 trillion cells

~23 thousand genes

…a hidden organ

Microbiome is defining in terms of human health

http://apc.ucc.ie

Intestinal Microbiota

Complex and dynamic community

1014 microorganisms/g contents

10 times more bacteria than human cells

100-fold more unique genes than our own genome

Important functions:

• Fermentation of food components• Production of SCFAs, vitamins, bioactives

• Barrier effect• Production of antimicrobial substances/pathogen inhibition

• Control of epithelial cell proliferation• Development of the immune system

METABOLIC

PROTECTIVE

MORPHOLOGIC

Guarner et al., 2011

http://apc.ucc.ie

Microbiota Impacts Host Metabolism

Ryan & Delzenne, 2016.

e.g. SCFA

http://apc.ucc.ie

Microbiome has intimate ties in almost every facet of host health

4

2

1

3

5

http://apc.ucc.ie

Gut Microbes and Obesity

Felix Sommer & Fredrik BäckhedNature Reviews Microbiology 11, 227-238 (April 2013)

http://apc.ucc.ie

Gut Microbes & Diabetes

http://apc.ucc.ie

Brain-Gut-Microbiota Axishttp://apc.ucc.ie

http://apc.ucc.ie

Human Gut Microbiota-large potential for health

• Necrotizing enterocolitis• Obesity• Diabetes • Metabolic syndrome• Irritable bowel syndrome • Inflammatory bowel disease•Colon cancer • Altered CNS development

PERTURBED MICROBIOTA

Bioactivemetabolites

http://apc.ucc.ie

GI microbiota over lifetimeYour microbiota assembles in the first years of life

and remains relatively stable until old age

Assembly Stability Collapse

Opportunities for interventions/diagnostics, for food, biotech & pharma

http://apc.ucc.iehttp://apc.ucc.ie

APC Hypothesis

• Diagnostics• Antibiotics• Anti-inflammatories• Medical foods• Functional foods

We can mine the gut

microbiota for products that

promote health or treat

disease

http://apc.ucc.ie

Mining the Gut Microbiota for Bioactives

http://apc.ucc.iehttp://apc.ucc.ie

Pharmabiotics

• Faecal transplants• Microbial consortia• Probiotics• Prebiotics• Metabolites• Bacteriophage

The gut microbiota

plays a role in determining

health - we can mine for

pharmabiotics

http://apc.ucc.ie

Microbiota Immune System

http://apc.ucc.ie

Modulation of Intestinal Microbiota

“Open window of opportunity” (Fouhy et al., 2012)

Edad temprana: Base de la homeostasis inmune para toda la vida

-9 meses Nacimiento ~ 36 meses

AlergiaObesidad

Enfermedades coronariasDiabetes

Enfermedades autoinmunes

SaludBajo riesgo de enfermedad

Kerperiem et al., 2012

Early life: Setting the immune balance for life

Allergy

Obesity

Coronary Heart

Diseases

Diabetes

Autoimmunity

HEALTHY LIFE

↓ Risk diseaseBirth ~ 36 months

-9 months

Importance of the correct establishment of intestinal microbiota

Early postnatal period constitutes key moment

Modulation to the establish a healthy microbial profile

http://apc.ucc.ie

Mode of Delivery: Phylum level

Hill et al (2017) (MICROBIOME)

http://apc.ucc.ie

Increase in Diversity over Time

Hill et al (2017) (MICROBIOME)

http://apc.ucc.ie

By Year 2 Microbiota Becomes More Adult-like

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Year 1 Year 2

Unclassified

Other

Clostridium_IV

Coprococcus

Alistipes

Erysipelotrichaceae incertae sedis

Clostridium sensu stricto

Gemmiger

Clostridium XI

Prevotella

Clostridium XVIII

Ruminococcus

Clostridium XlVa

Roseburia

Anaerostipes

Lachnospiracea incertae sedis

Blautia

Faecalibacterium

Bacteroides

Bifidobacterium

Shan

no

n D

iver

sity

Year 1 Year 2

23.9%

4.7%

d = 0.05

A)

B)

C)

*** *** (data unpublished)

http://apc.ucc.ie

Pharmabiotics

Gut Bacterial

Metabolites

Conjugated

Isomers

SCFA

NeuroactiveCompounds

Bioactive

PeptidesVitamin B

Exopolysaccharides

Bacteriocins

AntihypertensiveAntithrombiticCaseinophosphopeptidesImmunomodulatoryAntioxidantOpioid Cytomodulatory

http://apc.ucc.ie

Conjugated Linoleic Acid (CLA)

Anti-CancerImproves

Heart Health

Improves Immune System

Fat

Regulating

Improves Body Composition

Bone Formation

http://apc.ucc.ie

Recombinant Lb. paracasei producing t10, c12 CLA in vivo

t10, c12 CLA

LA

GLC

Lactoacillus paracasei

Expressing CLA isomerase (P. acnes)

Increased t10, c12 CLA content of adipose tissue following dietary intervention

0.00

0.01

0.02

0.03

***

g/1

00

g F

AM

E

VectorControl

CLAProducing

n = 8

8 weeks

Rosberg-Cody et al (2011) Microbiology

http://apc.ucc.ie

Production of Bioactive Lipids - CLA

%

Conversion

to CLA

90

80

70

60

50

49

30

20

10

LA

CLAGLC

Bifidobacterium breve

Bifidobacterium longum

http://apc.ucc.ie

r= -0.863

Linoleic acid

Linoleic acid + B. breve

c9, t11 CLA (g/100g FAME)

Lin

ole

ic a

cid (

g/1

00g F

AM

E)

Wall et al., AJCN 2009 89: 1393-1401

Administered B. breve is metabolically active in the gut

http://apc.ucc.ie

Impact of administered B. breve on FA composition

***

BALB/c mice SCID mice

LA

LA + B. breve

*

LA + B. breve

Control

*

Pigs

Wall et al., AJCN 2009, 2012

http://apc.ucc.ie

Elevated Lc-PUFA in Adipose Tissue and Brain

***

EPA (Eicosapentaenoic Acid)

*

DHA (Docosahexaenoic Acid)

n = 8

* = p<0.05*** = p<0.001

Unsupplemented

B. breve 702258 supplemented

Wall et al. Am. J. Clin. Nutr. 2009

http://apc.ucc.ie

Microbiome-mediated therapies for heart health?

Probiotic

Prebiotic

Archaebiotics

• BSH

• EPS

• Arabinoxylan

• Oat β-glucan

• TMA-Depletion

http://apc.ucc.ie

EPS Producing LAB: Probiotic for Heart Health?

EPS

http://apc.ucc.ie

Lb. mucosae 6426: Genome Sequence

Lactobacillus

mucosae DPC6426

The genome revealed a plethora of

homologues linked to carbohydrate

metabolism (EPS operon) and mucin

binding. In addition, several putative

bile salt hydrolase genes (BSH) were

identified, along with a potential novel

phage and CRISPR elements.

http://apc.ucc.ie

DPC6426 Reduces Atherosclerosis – In Vivo

n = 14n = 14n = 14

A 24-week intervention with either Live (lDPC) or Heat-Killed (hkDPC) Lb. mucosae DPC6426 fed in

conjunction with a high-fat diet significantly attenuated cholesterol (and in particular LDL-C) accumulation

when compared to the high-fat control (HFC). In turn, the strain reduced the development of aortic plaque

in the atherosclerosis-prone apolipoprotein-E-deficient (apo-E-/-) mouse model.

High Fat Control

Heat Killed

http://apc.ucc.ie

Bile Salt Hydrolase Activity & Specificity

Porcine Murine

(Ryan et al., unpublished)

http://apc.ucc.ie

Live Lactobacillus mucosae

Alters Microbiome Composition & Functionality

liveDPC

HFC

hkDPC

liveDPC

HFC

hkDPC

* *

**

16S MiSeq

U/w UniFrac

Faecal 1H-NMR OPLS-DA

(Ryan et al., unpublished)

http://apc.ucc.ie

Antiatherogenic Activity of DPC6426

http://apc.ucc.ie

Brain-gut-microbiota axis

• Brain gut axis is a bi-directional signalling between the brain and the GI tract (O’Mahony 2011; Dinan and Cryan, 2016)

• Gut microbiota may play a role in neuronal development and plasticity, modulating pain perception and even behaviour (Forsythe et al., 2010; Dinan

et al., Biol Psychiatry. 2013)

• Microbes produce a wide spectrum of neuroactive compounds, including fatty acids, GABA (Lyte, 2011; Barrett et al., 2012)

• Studies have shown the ability of probiotics to alleviate distress in humans and an anxiolytic-like activity in rats (Lyte, 2011)

http://apc.ucc.ie

Microbes and neurotransmitters

Genus Neurochemical

Lactobacillus, Bifidobacterium GABA

Streptococcus, Escherichia, Enterococcus,Lactococcus, Lactobacillus

Serotonin

Escherichia, Bacillus Norepinephrine

Escherichia, Bacillus, Lactococcus, Lactobacillus,Streptococcus

Dopamine

Lactobacillus, Bacillus Acetylcholine

Lactobacillus, Lactococcus, Streptococcus, Enterococcus

Histamine

From Wall et al., 2014; Bacterial Neuroactive Compounds Produced by Psychobiotics Adv Exp Med & Biol 817, pp 221-241

http://apc.ucc.ie

GABA = Microbial Metabolite

• γ-aminobutyric acid (GABA) is synthesised through decarboxylation of glutamic acid by glutamic acid decarboxylase (GAD)

• GABA: main inhibitory neurotransmitter in the CNS-biological functions of GABA are mediated by activation of GABA receptors in the brain and periphery

91 intestinally-derived bacteria assessed, one Lactobacillus brevis strain (Lb. brevis DPC 6108) could convert up to 90% MSG to GABA in vitro

http://apc.ucc.ie

Anti-anxiety effect of GABA producing Lb brevis 6108

*P<0.05

0.0511

Inner Zone distance moved

0

100

200

300

400

500

600

UnsupplementedGABA powderL. brevis DPC 6108

cm

Inner Zone duration

0

10

20

30

40

50

sec

Total distance moved

0

1000

2000

3000

4000

5000

cm

*

(Marques et al., unpublished)

http://apc.ucc.ie

Sunday 28th June 2015

http://apc.ucc.ie

Gut Microbes, Metabolites and Health

• Gut Microbiome-central role in host metabolism

• Microbial metabolism-range of signalling molecules, which directly and indirectly impact on host lipid metabolism/cholesterol synthesis/brain function

• Probiotics have potential, but understanding mechanisms critical

• Future studies required to fully comprehend role of gut microbiome in: host-lipid metabolism, CNS development, ageing

• Targeted gut microbiome modulation dietary interventions for optimised health throughout life

http://apc.ucc.ie

Acknowledgments

Paul RossPaul O’TooleGer FitzgeraldFergus ShanahanTed DinanJohn CryanNoel CapliceEamonn QuigleyTony RyanGene Dempsey

RESEARCHERS

Paul RyanRebecca WallElaine PattersonRuairi RobertsonLis LondonTatiana MarquesMairead CoakleyEva RosbergAlan HennessyEoin Barrett

COLLABORATORS