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Group 3

Bioactive compounds : the Grand Canyon of our field

How gut microbiota establish the dialogue with the host, namely upon treatment with

probiotics and prebiotics

New targets, models and methodology

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Our contributors

• Speakers : George Fahey, Eileen Murphy, Fredrik Backhed, Karen Scott, Jon Swann, Patrice Cani, Carissa Thomas and Delphine Saulnier, Wendy Russell, Sandrine Claus, Catherine Stanton, Claire Merrifield, Bob Rastall

• Other attendees : Lori Lathrop Stern, JoMay Chow, Maeve Murphy, Barbara Miller, Douwina Bosscher, Agnes Meheus, Sarah Meeuws, Cathy Lichtenwald, Bo Möllstam, Mike Russell, Reg Fletcher, Agnes Meheust

• Chairs: Glenn Gibson and Nathalie Delzenne

LPS

3

Proglucagon mRNA

?

GLP2

LPS Stress

eCB eCBGPR43

SCFA

LPS

4

Food

Small intestine large intestine

Digestion ofstarches, sugars, fat, protein

Fate of ingested dietary products

Microbial fermentation

oligosaccharides, RS, Fibre (NSP),

SCFA, phytochemicals,

other metabolites, minerals

Blood stream

faeces

Mono-saccharides, fat, aminoacids

absorption

Undigested

carbohydrate,

Lignin, unabsorbed

Nutrients, SCFA

Proximal Distal

[Adapted from Topping & Clifton, 2001Physiol Rev. 81; 1031-1064] SCFA concentration gradient

Acetate (3)Transported to peripheral tissues,

used in triglyceride/cholesterol synthesis

Propionate (1)Used in gluconeogenesis

in liver

Butyrate (1)Energy source for

colonocytes

0

10

20

30

40

50

60

70

80

90

Acetate Propionate Butyrate Isobutyrate Isovalerate

Fae

cal s

hort

-cha

in f

atty

aci

ds [m

M]

Diet:

Maintenance

Moderate carbohydrate

Low carbohydrate

Significance -

A, B, C: P < 0.05

A

AA

B

B

B

B

B

C

[Duncan et al (2007) AEM]

Production of SCFA as dietary carbohydrate

Human study - Effect of different dietary carbohydrate levels on

detection of faecal short-chain fatty acids

5

% bacterial (Eub338) count in faeces

0

5

10

15

20

25

30

35

40

Bacterial group

% E

ub33

8

Diet:

Maintenance

Moderate carbohydrate

Low carbohydrate

A, B, C: P < 0.05

Detection by fluorescent in situ hybridisation (FISH)

A

BB

[Duncan et al (2007) AEM]

Response of bacterial groups to dietary change

A

C

B

C

A

B

SCFA

CLA

LPS

6

Linoleic Acid [C18:2 c9, c12]

CLA [c18:2 c9, t11]

CLA [C18:2 t10, c12]

CLA [C18:2 t9, t11]

Screening of Human Gut Microbes for Conjugated Linoleic Acid (CLA) Production

B. breve

CLA

%Conversionto CLA

Production of Bioactive Lipids by Commensals

90

80

70

60

50

49

30

20

10

LA

CLAGLC

Rosbery-Cody et al., AEM 2004Barrett et al. AEM 2007Hennessy at al. JAM 2009

CLA Producing Bifidobacterium

7

0

30

60

90

120

0 1 2 5 10

t 9, t 11 CLA (u g/mL)

cell

viab

ility

(%

) c9, t11 CLA

Statistical analysis: * p<0.05; ** p<0.01; *** p<0.001

COOHc9, t11 CLA

t9, t11 CLA

Statistical analysis: * p<0.05; ** p<0.01; *** p<0.001

COOHt9, t11 CLA

0

30

60

90

120

0 5 10 15 20

c 9, t 11 CLA (u g/mL)

cell

viab

ility

(%

)48 hr incubation 4 day incubation

48 hr incubation 4 day incubation

t9, t11 CLA appears to have a more potent anti-proliferative effect than the c9, t11 CLA isomer using SW480 colon cancer cells

****

***

**

*

*****

**

Coakley et al. Nutr. Cancer 2006

SCFA

CLA

Bile acids

LPS

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Germ-free bile acid signature skewed towards tauro-conjugates

Same trend is also observed following bacterial attenuation via antibiotic

administration

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Functional and signaling changes associated with bile acids in the liver

SCFA

CLA

Bile acids

Immunomodulins

LPS

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Hypothesis: Secreted Factors from L. reuteri ATCC PTA 6475 Inhibit TNF Production by Modulating Inflammatory Signaling Pathways

TLR2 agonist (PCK)

+

Secreted factors (<3kDa)

from Lactobacillus reuteri

Increased TNF

Decreased TNF

Human myeloid cells

� Folate is present in the gastrointestinal tract as f olic acid and folylpolyglutamates

� Recent studies show that probiotics producing folate can improve folate status (Mohammad et al, 2006; Strozzi & Mogna, 2008)

� L. reuteri is a folate producer (Wegkamp et al, 2008)

Focus on Essential Bacterial Molecules

Produced by L. reuteri

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Cell Pellet Washes of ATCC PTA 6475 & ATCC 6475∆fpgs2 Inhibit TNF Production

56

% (

10

mg

/ml)

62

% (

10

mg

/ml)

77

% (

10

mg

/ml)

71

% (

10

mg

/ml)

94

% (

LDM

III)

ControlsAnaerobic Aerobic

� ATCC 6475Δfpgs1 loses the ability to inhibit TNF – no folylpolyglutamates seen on MALDI-MS

� ATCC 6475Δfpgs2 maintains ability to inhibit TNF – large folate peak seen on MALDI-MS

� No TNF inhibition by DSM17938

Cell Surface Assoc. Factors

SCFA

CLA

Bile acids

Immunomodulins

Anti-inflammatory metabolites

LPS

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Ferulic Acid - BioactivityProtective: Cardiovascular Disease, Cancer, Dementia,

Diabetes, Irradiation, Immune Disorders, IBD

Redox Activity0.25-0.11

-0.08

0.23

-0.07

0.08

(0.70)

Russell et al Bioorganic & Medicinal Chemistry 2005 13:2537-2546

Potent anti-oxidantFree radical scavenging propertiesAttenuates oxidative stressChain-breaking anti-oxidantInhibits LDL oxidation

Inflammatory Activity

Russell et al BBA Molecular Basis of Disease 2006 1762/1:124-130

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************

*********

20

40

60

80

100

120

140

Inhi

biti

on/E

nhan

cem

ent

of

pros

tano

idpr

oduc

tion

(%

) Inhibition

Enhancement

vanillin sinapicacid

eugenol cinnamicacid

vanillicacid

ferulicacid

caffeicacidn

curcumin 4-hydroxybenzoic acid

salicylicacid

proto-catechuic

gentisicacid

ICAM-1, VCAM-1, ERK, JNK, MAPK, TNF- αααα, IL-1ββββ, NF-kB, IL-10 Akt, RAS, Ets2, Wee 1, COX-2, MMP-1, IL-6, IFN-γγγγ, PGE2, iNOS, NIK/IKK

Ferulic Acid – Colonic Bioavailability

OOH

OH

O

OOH

OH

O

OOH

OH

OH

OOH

OH

OOH

OH

O

OOH

OH

O

OOH

OH

OH

OOH

OH

Russell et al Nutrition and Cancer 2008 60/5:636-642

01234567

89

10

Ferulic Acid Met. 1 Met. 2 Met. 3

Con

cent

ratio

n (u

mol

dm

-3) Ferulic Acid Content

in Faecal Watersof Free Living Individuals

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Ferulic Metabolites – Effect of Carbohydrate

n = 14; male; age 54 +/- 13 years; BMI > 27; NSP/RS cross-over

0

20

40

60

80

100

120

140

Ferulic Acid Met. 1 Met. 2 Met. 3

Con

cent

ratio

n (u

g cm

-3) Non Starch Polysaccharide Diet

Resistant Starch Diet

P < 0.01P < 0.01

P < 0.01Maintenance diet

Ferulic Acid – Microbial Metabolism

de-esterification hydrogenation demethylation dehydroxylation

OOH

OH

O

OOH

OH

O

OOH

OH

OH

OOH

OH

OO

OH

O

OOH

OH

O

OOH

OH

O

OOH

OH

OH

OOH

OH

OO

OH

O

Increase feruloyl esterase activity = Increase ferulic acid

Decrease hydrogenase, demethylase and dehydroxylase activity?

51% 25% 56% 66%

Inhibition of neoplastic prostanoids

Russell et al Nutrition and Cancer 2008 60/5:636-642

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SCFA

CLA

Bile acids

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

LPS

To assess the effect of the bacteriocin-producing probiotic strain Lactobacillus salivarius UCC118 (UCC118 Bac+) and a non-bacteriocin-producing derivative (UCC118 Bac-) on the gut microbiota in vivo and

impact on obesity.

AIM

� Bacteriocins are a heterogeneous family of anti-microbials produced by many bacterial species.

�Due to their anti-microbial activity, bacteriocins may play a significant role in determining dominance and flux in gut populations, and could potentially be manipulated to drive the development of a ‘healthy’ microbiota

�First report to directly assess the effect of a bacteriocin on the gut microbiota in vivo

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10% LF diet

45% HF diet

45% HF diet

45% HF diet

(12 weeks)Intervention

(8 weeks)

Lean DIO

* DIO + UCC118 Bac +

**DIO + UCC118 Bac -

Markers of energy harvestFecal Energy SCFA

Metabolic HealthFood IntakeBody weight/ compositionTissue weightsPlasma Glucose, Insulin, TNF-α, TG, Liver TG

Pyrosequencing

Groups (n=10)

* Bac+ = bacteriocin producer**Bac-= non -bacteriocin producer

Experimental design

10% LF diet

45% HF diet

45% HF diet

45% HF diet

(12 weeks)Intervention

(8 weeks)

Lean DIO

* DIO + UCC118 Bac +

**DIO + UCC118 Bac -

Markers of energy harvestFecal Energy SCFA

Pyrosequencing

Groups (n=10)

* Bac+ = bacteriocin**Bac

Lb. salivarius UCC118 DIO study

29Dr. Paul Cotter, Teagasc

45% HF diet DIO + vancomycin

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UCC118 and bacterocin alters the composition of the gut microbiota in diet-induced obesity.

Bacteriocin prodn: ↓ Actinobacteria ↑ Bacteroidetes & Proteobacteria

=

DIO + UCC118Bac -

1.1%

0.1%

0.4%3.4%

1.1%17.6%

0.1%

72.4%

5.2%

0.4%3.4%

Proteobacteria

Bacteriodetes

SpirochaetesFirmicutes

Actinobacteria

Tenericutes

Deferribacteres

DIO + UCC118 Bac+

1.6%

23.9%

66.9%

1.5%

6.1% 1.6%6.1%

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SCFA

CLA

Bile acids

Probiotics

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

LPS

SCFA

CLA

Bile acids

Probiotics

Prebiotics

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

LPS

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• Carbohydrate co-product of fiber board manufacturing

• Produced by Temple Inland, Diboll, TX

Temulose

Loblolly Pine

Chemical Structure• Mixture of galactoglucomannans and arabinoxylans

• Mannose:glucose:galactose ratio – 6:2:1

− β-1,4 linkage between mannose and glucose

− ɑ-1,6 linkage between mannose and galactose

• Degree of polymerization of 2-50 units

Arabinoxylan

Galactoglucomannan oligosaccharide

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Short-chain Fatty Acids

0

50

100

150

200

250

300

350

400

Acetate Propionate Butyrate Total SCFA

µmo

l/g D

M

ScFOS Safmannan Temulose molasses TBS GL GM GS

Means without a common superscript letter differ (P < 0.05).

b

a

d d

c

d d

b

a

d c

fe

c

ab b b b c d

b

a

d

c

fe

d

4

5

6

7

8

9

10

11

Bifidobacterium spp. Lactobacillus spp. E. coli C. perfringens

CF

U,

log

10/m

l

ScFOS Safmannan Temulose molasses TBS GL GM GS

Microbial Concentration

Means without a common superscript letter differ (P < 0.05).

bc

a

ed

b b cd

aba

bab a abab

c c bc bca a ab

bca

cabab

bcab

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SCFA

CLA

Bile acids

Probiotics Prebiotics

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

LPS

Inhibition of cholera toxin by GOS

NeuNAcαααα2↓↓↓↓6

Galββββ1→→→→3GalNAcββββ1→→→→4Galββββ1→→→→4Glc

Cellular receptor is ganglioside GM1

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Galββββ1→4Galββββ1→4Glc Galββββ1→3Galββββ1→4Glc

NeuNAcαααα2↓↓↓↓6

Galββββ1→→→→3GlcNAcββββ1→→→→4Galββββ1→→→→4Glc

GOS components

GM1

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Inhibition of cholera toxin by GOS

0102030405060708090

100

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

GOS iex fraction (100mg/ml)

% I

nhib

itio

n

0

10

20

30

40

50

60

EC

50(m

g/m

l)

Sinclair et al (2009) Journal of Agricultural and Food

Chemistry 57: 3113-3119 Competitive ELISA assay

SCFA

CLA

Bile acids

Probiotics Prebiotics

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

LPS

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Approach

Identifying bacteria present in humans diseases

Identify their origin

Establish model to test causality

Test Koch’s postulate

Atherosclerosis as an example

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Distinct microbial profile in the plaque

SCFA

CLA

Bile acids

Probiotics Prebiotics

Immunomodulins

Anti-inflammatory metabolites

Bacteriocins

Metabolites

LPS

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Dietary intervention in the weaning pig - Experimental Protocol – N=7

Aimso To establish if B.lactis has an effect on the pig which is detectable by metabonomics

o To observe the response of B.lactis to different weaning diets

o Do metabolic profiles correlate with other experimental immune parameters?

Weaning Diet induces a Sustainable Metabolic Reprogramming Event

Greatest metabolic variation is due to

initial weaning diet despite 4 week

washout period, this result implies a

‘Sustainable Metabolic Reprogramming

Event’ that occurs during the weaning

period.

Urinary Metabolic Signature

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49

USE OF ANTIBIOTICS TO RESHAPE THE GUT FLORA

• 3 groups of mice (n=6):

– Control group

– Bacterially suppressed group (8 days of antibiotics)

– Recolonised group (4 days of antibiotics + 4 days of

washout)

• Streptomycin/Penicillin in drinking water

• 1H NMR spectroscopy of urine collected at day 8

• FISH analysis of faeces collected at day 8

Suppressed

Control

50microbiota and influence the host metabolism

Swann J (personal communication)

FISH analysis of intestinal contents at day 8

Metabolic profiling analysis

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What next?

• An ISAPP meeting dedicated to bioactive compounds!

• What are the possibilities for industry in exploiting the bioactives identified in the gut functional ecosystem?

• New model systems and systems biology approaches will give new insights into the functional ecology of the gut