Post on 29-May-2019
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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
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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
21
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
25
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