El 29 de marzo de 2016 celebramos un Simposio Internacional sobre el 'Impacto de las ciencias ómicas en la medicina, nutrición y biotecnología'. Organizado por la Fundación Ramón Areces en colaboración con la Real Academia Nacional de Medicina y BioEuroLatina, abordó cómo un mejor conocimiento del genoma humano está permitiendo notables avances hacia una medicina de precisión.

1. MICROBIOME FOR HUMAN HEALTH
LESSONS OF A METAGENOMIC SCAN
« IMPACT OF OMIC SCIENCES IN
MEDICINE, NUTRITION AND BIOTECHNOLOGY »
MADRID, MARCH 29TH 2016
Hervé M. Blottière
FInE lab « Functionality of the Intestinal Ecosystem»,
Micalis Institute, INRA, AgroParisTech, Université Paris-Saclay
&
MetaGenoPolis
78350 Jouy en Josas

2. « People are not just people. They are an
awful lot of microbes, too »
ME, MYSELF, US
The Economist , August 18th 2012

3. THE HUMAN INTESTINAL MICROBIOTA
Faecalibacterium prausnitzii Ruminococcus spp Clostridium difficile
From mice cecumPhotos INRA
Bacteroides dorei Escherichia coliBacteria anchored in a
Peyer patch
Mouse intestine
 100 trillion microorganisms ;
 10 times the number of cells in the human body;
 Hundreds of different species predominantly not yet cultured (~70% of
dominant species);
 a few dozen conserved between individuals (core); a stable community.
 A true organ ; geared to « protecting our health and well-
being »« throughout all stages of our life »
 A key organ, interacting with food (fermentation,…); interacting with our
cells (Immune & nervous systems,…); protecting against pathogens
(barrier function);…
3,9·1013 en 2016
x 1.3

4. Microbiota as an asset
 Utilisation of indigestible
carbohydrates
 Epithelial nutrients (e.g. SCFAs)
 Synthesis of B & K vitamins
 Conversion of prodrugs
 Metabolism of dietary carcinogens
 Control motility/Peristalsis
 Defenses - bacterial antagonism
 Priming of mucosal immunity
Microbiota as a liability
 Procarcinogens  carcinogens
 Overgrowth syndromes
 Opportunism - Translocation
 Essential ingredient for IBD
 Implicated in obesity, metabolic
syndrome and colorectal cancer
Globally conserved :
With a few exceptions :
- Microbial metabolites - Methanogenesis
- Microbial enzymes- cholesterol reduction
- Barrier effect - lignan metabolism
- polyphenol metabolism
FUNCTIONS OF THE INTESTINAL MICROBIOTA
barrier
Physio
defensesIntestinal
Microbiota
Nutrition

5. METAGENOME : genomes of all dominant microbes in an ecosystem
Whole
Genome
Shotgun
sequencing
Assembly and annotation
Reference gene catalog
and gene counts
A technical revolution since the turn of the century
DNA
extraction
Lessons from early human intestinal tract metagenomics:
• Qin Nature 2010 => 3.3 million gene catalog ; both core metagenome & rare genes
• Arumugam Nature 2011 => 3 enterotypes, preferred ecological arrangements
• Schloissnig Nature 2012 => stability at SNPs level (strains)
• Qin Nature 2012 => metagenomic dysbiosis in T2D
• Le Chatelier Nature 2013 => metagenomic dysbiosis in obesity ; diagnostic signatures
• Cotillard Nature 2013 => metagenomic dysbiosis in obesity ; low gene count as stratifier
• Qin Nature 2014 => metagenomic dysbiosis in liver cirrhosis
• Li Nat Biotech 2014 => 10 million gene catalog ; core metagenome unchanged
• Nielsen Nat Biotech 2014 => co-abundant gene clustering and metagenomic species
• Xiao Nat Biotech 2015 => Mouse gene catalog ; environment dependent
• Shoaie Cell Metabol 2015 => nutrition and intestinal metabolome
• Forslund Nature 2015 => Metformin signature in T2D

6. A REFERENCE CATALOG OF 10 MILLION GUT GENES
Rare genes are
increasing
Common genes are not
Individuals from MetaHIT, Chinese and HMP studies, n=1267
Li et al. Nature Biotech, 2014
124 MetaHIT subjects
3.3 million gut genes
Qin, Nature 2010
Total
>5%
>10%
>20%
>50%
3.3

7. HUMANS SHARE A CORE MICROBIOME, AND YET DIFFER
AT THE METAGENOMIC LEVEL
On average, each individual carries ~540 000 genes
of the initial 3.3 million genes catalog
Similarity:
Core metagenome genes :
~50 % of an individual’s genes are
shared by at least 50 % of
individuals of the cohort
We are all rather similar ! but not identical!!
Yet, individuality:
Rare genes :
genes shared by less than 20 %
of individuals
= 2.4 million genes
Qin, Raes et al, Nature 2010
Mouse catalogue : Xiao et al, Nature Biotech, 2015
Very different from mice : 4% shared genes in the 2 catalogues

8. HUMANS MICROBIOMES DIFFER AT THE LEVEL OF
ECOLOGICAL ARRANGEMENT : THE ENTEROTYPES
In a attempt to characterize the
intestinal microbiome of ‘the average
human’, we observed
…
an organization of intestinal
metagenomes in 3 assemblages of
genes and microbial taxa :
order in chaos !?!
Qin et al, Nature 2010
Arumugam, Raes et al, Nature 2011
May 2011
the enterotypes

9. Ecology underlying enterotypes should be better
understood
Bacteroides Prevotella Ruminococcus
Impact of diet ?
Wu Science 2011
Tap EM 2015

10. HUMAN GUT MICROBIOMES DIFFER AT THE LEVEL
OF
GENE RICHNESS (DIVERSITY)
n=277
High gene
count
Low gene
count
Le Chatelier, et al, Nature 2013; Cottillard et al, Nature, 2013
LGC
≈15 % of lean
individuals
≈ 25%
Overweight-
moderate obese
≈ 75%
Morbid obese
before bypass
surgery
68 « species » significantly linked to gene count

11. Nielsen, Almeida, et al. Nature Biotech, 2014
GENE CATALOG CLUSTERED IN METAGENOMIC UNITS BY
CO-ABUNDANCE BINNING
MGS
MGU
Interaction network
CAG: Co-abundant Gene Group
• All genes of a species should have the same frequency in an individual
• Although proportions of a species vary greatly between individuals
• Genes of a species will precisely co-vary in abundance in each
741 large MGU (>700 Genes) correspond to bacterial species
(MetaGenomic Species; 85% previously unknown)
238 high quality genomes reconstructed
6640 small MGU (phages, plasmids, CRISPR…)

12. QUANTITATIVE METAGENOMIC PIPELINE AT
METAGENOPOLIS:
SAMBO-METAQUANT-INFOBIOSTAT
sample
collection
sequencing reference
construction
gene profiling
sequence mapping
onto gene catalogue
stool sample
total DNA
library preparation
SOLiD/Illumina/Proton
sequencing
short sequences
30-50 million
reference gene
catalogue
gene counts
genes
bioinformatics
/statistics analyses
preprocessing /
normalization &
dimension reduction
relation with
clinical data
Identify clinically
relevant groups
build and test
prediction models
catalogue
structuration
individuals
1
2
3
4
1 2 3 4
catalogue
annotation
MetaHIT & iMOMi Databases
• 4 - 8 million genes
• 6000 + genomes
Standardization is critical
http://www.microbiome-standards.org

13. Can the neglected organ inform on a risk of chronic diseases?
Can it be a target for intervention?
CHRONIC DISEASES INCREASE STEADILY IN
INDUSTRIALIZED COUNTRIES
Bach JF, N Eng J Med 2002

14. Crohn Seksik, 2003; Sokol, 2006, 2008, 2009; Mondot 2010
Ulcerative colitis Sokol, 2008; Martinez, 2008; Lepage 2011
IBS Rajilic-Stojanovic 2011; Saulnier 2011; Chassard 2012;
Pouchitis Lim, 2009; Kühbacher, 2006
Obesity Ley, 2007; Furet, 2011, Le Chatelier, 2013; Cotillard, 2013
Type-2 diabetes Cani and Delzenne, 2009; Burcelin 2011, Qin, 2012
Type-1 diabetes Dessein, 2009; Wen, 2008
Coeliac disease Nadal, 2007; Collado, 2009
Allergy Björkstén 2009; Abrahamsson 2012, Russel 2012
Autism Finegold, 2002; Paracho, 2005
Colorectal cancer Mai, 2007; Scanlan, 2008; Sobhani 2011
Frailty in seniors Van Tongeren, 2005, Claessen et al., 2012
Breast cancer Velicer et al., 2004
HIV infection Gori, 2008
Liver Henao-Mejia 2012; Qin 2014
Cardiovascular Wang 2011; Karlsson 2012, Metacardis EU project
Other….
PERTURBATION OF INTESTINAL MICROBIOTA
AS A POSSIBLE CHRONIC DISEASE FACTOR
Indications from metagenomic clinical studies
Dysbiosis :
 Loss of keystone species
 Loss of richness
 Increased pathobionts
 Metabolic shift

15. METAGENOMIC SIGNATURES OF DYSBIOSIS
IN IMMUNE DISEASES
Bacterial genes and genomes specific of the microbiome of
patients
BMI
Inflammatory bowel diseases and obesity
0.10.1
generans
nalis.M50.1
f ormis
occus.comes.SL7.1
dium.sp.SS2.1
ubacterium.rectale.M104.1
anisolv ens.XB1A
5
gum.subsp..inf antis.CCUG.52486
es.merdae
olescentis
coccus.torques.L2.14
p..D1
eroides.distasonis.ATCC.8503
ccus.obeum.A2.162
us.bromii.L2.63
cae
.thermophilus.LMD.9
cola
ium.v entriosum
ccus.lactaris
pillosusL2.50
ulum.v ariabile
v ibacter.smithii.DSM2375
us.colihominis
1
.pamelaeae.gen.nov .sp.Nov
C.BAA.835
ctus
rof orme
prophilus
H1
842
rev e
mutans.UA159
5586
mentum.IFO.3956es.odontoly ticus
uinis.SK36
AA.381
antella.f ormatexigens
y riv ibrio.f ibrisolv ens.16.4
a.stadtmanae.DSM.3091
aeruginosa.LESB58
.subsp..multocida.str..Pm70
senteroides.subsp..mesenteroides.ATCC.8293
oncisus.13826
ndidatus.Sulcia.muelleri.GWSS
ardii.ATCC.8290
eri.SD2112
P76
is.stercorihominis
idium.phy tof ermentans.ISDg
nensis.DSM.16047eticus.DPC.4571
TCC.25302us.ATCC.15305
generans
nalis.M50.1
f ormis
occus.comes.SL7.1
dium.sp.SS2.1
ubacterium.rectale.M104.1
tena
anisolv ens.XB1A
5
gum.subsp..inf antis.CCUG.52486
m.hallii
es.merdae
olescentis
coccus.torques.L2.14
p..D1
eroides.distasonis.ATCC.8503
.siraeum.70.3
ccus.obeum.A2.162
us.bromii.L2.63
cae
.thermophilus.LMD.9
cola
ium.v entriosum
usccus.lactaris
pillosusL2.50
ulum.v ariabile
v ibacter.smithii.DSM2375
us.colihominis
1
.pamelaeae.gen.nov .sp.Nov
ansia.muciniphila.ATCC.BAA.835
ctus
rof orme
prophilus
H1
842
densssotus
rev e
mutans.UA159
5586
mentum.IFO.3956es.odontoly ticus
uinis.SK36
cter.hominis.ATCC.BAA.381
antella.f ormatexigens
y riv ibrio.f ibrisolv ens.16.4
a.stadtmanae.DSM.3091
aeruginosa.LESB58
is.SH0165
.subsp..multocida.str..Pm70
senteroides.subsp..mesenteroides.ATCC.8293
oncisus.13826
plei.str..Twist
ndidatus.Sulcia.muelleri.GWSS
ardii.ATCC.8290
eri.SD2112
P76
is.stercorihominis
idium.phy tof ermentans.ISDg
nensis.DSM.16047eticus.DPC.4571
nsonii.NCC.533aracasei.subsp..paracasei.ATCC.25302saprophy ticus.subsp..saprophy ticus.ATCC.15305
s
d = 5
Scores and classes
N:N
Y:CD
Y:UC
Axis3
d = 2
Y:UC
p-value: 0.031
Crohn
Patients
UC
Patients
Healthy
Controls
Guarner (HUVH, Barcelona)
Wang Jun (BGI, Shenzen)
Ehrlich, Lepage, Tap (INRA)
Pedersen (SDC, Copenhagen)
Wang Jun (BGI, Shenzen)
Ehrlich (INRA, Paris)

16. 12 MGS
AUC = 0.84
Linear additive model
Metagenomic species show a good discrimination power between
obese and lean, in contrast to human genome
ROCs for
individual
MGS
False positive
True positive
INTESTINAL MICROBIOTA AND OBESITY IN HUMAN
n= 154 Danes
32 obesity risk loci
AUC = 0.58
False positive rate
n = 8,120
European
Speliotes et al. Nature Genetics 2010
Le chatelier et al, Nature, 2013

17. Le Chatelier et al, Nature 2013
Low to High
gene count (French or Danes)
Known
species
n=10
Unknown
species
n=48
Signature species (n= 58)
ROC analysis
4 species
LGC HGC
In overweight & obesity,
Low Gene Count (low bacterial richness) individuals
have less healthy metabolic & inflammatory traits:
MICROBIOTA GENE COUNT / DIVERSITY IS
A HEALTH-ASSOCIATED STRATIFIER
Increased adiposity, insulin resistance, dyslipidaemia,
inflammation, that predispose for type 2 diabetes, cardio-
vascular disease, cancer

18. FUNCTIONAL SHIFTS IN THE LGC MICROBIOME
Le chatelier et al, Nature, 2013
SCFA
Butyrate
• Energy source for IEC
• Trophic & barrier functions
• HDAC inhibitor (gene regulation)
• GPR agonist – PYY & GLP1
• Immunomodulatory effects
Blottière et al, PNS 2003;
Segain et al, Gut, 2000

19. intervention : high protein, low fat and
low glycemic index carb with a highly
diverse fibre content (1200 to 1500 Kcal)
6 w-intervention 6w-stabilisation
Low gene
count
High gene count
DIETARY INTERVENTION AND MICROBIOTA RICHNESS
Low gene count individuals lose less weight in response to diet
+ 25%
Cotillard et al, Nature 2013
In low-gene count microbiota, a high-diverse fiber supply may promote an
increase in dominant microbiota gene richness

20. DIETARY HABITS IMPACT GENE RICHNESS
3 dietary patterns are determined based on food frequency
records, using 7days-means for 26 food categories
Cluster 2
Cluster 3
Cluster 1
‘richness in fibres’Canonical discriminant analysis
Healthier’ dietary habits are associated with higher gene richness
Kong et al, PLoS one 2014
yoghurt
water
vegetables
fruits
soups
Potatoes
including chips
sweets
Sweetened soft drinks
- +
Karine Clément, ICAN,
& Danone Research .

21. DIETARY MODULATION OF GUT MICROBIOTA CONTRIBUTES TO
ALLEVIATION OF BOTH GENETIC AND SIMPLE OBESITY IN CHILDREN
diet based on whole-grains, traditional Chinese medicinal foods and
prebiotics
Phenotype
improved
Microbiota
modulated
PWS Prader Willi Syndrome
SO Simple Obesity
Zhang C-H et al, eBioMedicine, 2015
Functional level
Composition

22. Tremaroli et Bäckhed, Nature 2012; McFall-
Ngai et al, 2013
Brain
Liver
Adipose tissue
Muscle
Intestine
Microbiote
MECHANISMS LINKING DYSBIOSIS AND
DISEASES

23. Functional Metagenomics
 The intestinal microbiota plays a key role on Human
Health, but how ?
 Few bacterial compounds/metabolites modulating
host cell response identified
 An objective : identify bacterial compounds/genes
with functional interest and potential applications in
the gut microbiota
 Mining the microbiome for different functions
 How to study these interactions when 80 % of the
commensals are not yet cultured ?

24. Bacterial fraction
Large fragment (40 kb)
Metagenomic DNA
DNA extraction
Fosmid vector
cloning
Transformation
in E.coli
Metagenomic
Library in E.coli
Identification of
• clones
• genes
• enzymes
• molecules
HTS
High throughput
screening
Reporter Human
Intestinal Epithelial Cells
Substrate of interest
(fibers/carbohydrates)
Enzymatic activities (hydrolyse)
Reporter gene activities
Ecosystem (feces)
FUNCTIONAL METAGENOMICS
Gloux et al., AEM, 2007
Lakhdari et al., Pone, 2010; de Wouters et al., Pone, 2014

25. ROBOTIC EQUIPMENTS FOR HTS
Castor 1 & 2 – Human cells
Pollux 1 & 2 – Bacteria culture
Development of MetaFun

26. Metagenomiclibraries
Identification of
• Clones
• Genes
• Molecules
Sampling &
DNA
extraction
Cloning,
recombinants
selection
Homogenous
DNA library
Screening
10 libraries = 340 000
clones
→ 13 million genes
HTS
Reporter human
intestinal epithelial
cells
FUNCTIONAL METAGENOMICS TO
STUDY MICROBIOTA – HOST
CROSSTALK

27. Endocrine Metabolism Proliferation Immunity
TSLP, TGF-β, IDO,
PlgR, RALDH1,…
Muc2,
Cyclin D1
Fiaf/Angptl4PYY, GLP-1
Focusing on functional bacterial genes…
… and metabolic pathways
NF-κB, AP1, AhRAP1, p53PPARγCa2+
Epithelial cell
Enteroendocrine cell
TARGETED INTESTINAL FUNCTIONS

28. HTS OF METAGENOMIC CLONES
Lakhdari et al, PLoS One, 2010
stimulators
inhibitors
Growth of metagenomic clones (DO 600nm)

29. AN EXEMPLE : CLONE 52B7
(FROM CROHN ‘S DISEASE LIBRARY)
Random transposon mutagenesis :
*52B7/A and C : transposon targeted the same gene
encoding an ABC transporter permease
*52B7/B transposon targeted a putative lipoprotein with
unknown function.
the 2 targeted genes code for : a lipoprotein
and a lipoprotein transporter
Lakhdari et al, PLoS one, 2010
Stimulates NF-kB in several cell lineages and IL-8 secretion in IEC
Secreted in the supernatant, size 50 KD, Trypsin sensitive
From Bacteroides vulgatus

30. 5 clones were identified and selected after a
screening of 15,000 metagenomic clones from a
healthy donor library
Cellular reporter systems used: TGFb and IDO
promoter assay in HT-29 cells.
Work in progress , joint scientific program with Enterome. Aiming to:
• Identify, purify the bacterial components and elucidate their structures
• decipher the activation pathway, receptors involved
• Validate bacterial components on human cellular models (in vitro) or mice models (in
vivo)
Activation via NF-kB. MyD88 and TLR
independent pathway.
Sequences from the selected clones belong to
Firmicutes / Clostridia XIVa / Ruminococcus.sp
Control ControlClone Clone
Nicolas Lapaque et al, on going work
OTHER EXEMPLE: MODULATION OF IDO/TGF EXPRESSION

31. TAKE HOME MESSAGES
 The Human Gut metagenome is much more complex than
expected. 10 million genes facing 23 000 genes;
 Although most gut bacteria are still not cultured, MGS can be
reconstituted from metagenomic sequencing;
 Humans share a core microbiome and yet they differ by
genes, species, enterotypes (ecology) and gene count
(microbiota diversity).
 Metagenomic revealed pathology- associated species;
 Functional metagenomic reveals new bacterial genes involved
in the cross-talk with human cells;
 Human Gut Metagenome is a powerful reservoir of bioactive
compounds.

32. Nicolas Lapaque,
Catherine Juste,
Christel Maillet-Béra
Jean-Marc Lelièvre
et al….
Joël Doré
« Functionality of the Intestinal Ecosystem » FInE/Blottière lab,
INRA UMR 1319 Micalis, Jouy en Josas
Karine Clément (ICAN, CHU Pitié Salpétrière)
& MetaCardis Consortium
Harry Sokol (Hôpital Saint Antoine)
Sven Pettersson et col. (Karolinska Institute)
Maria Rescigno (IEO, Milan)
Oluf Pederson (Novo Nordisk Fundation, Copenhague)
Francisco Guarner (Val Hebron Hosp., Barcelone)
S. Dusko Ehrlich
& MetaHIT Consortium
Florence Haimet
Nicolas Pons
Emmanuelle Le Chatelier
Véronique Lejard
Florence Levenez
Jean-Michel Batto
et al….