Pr. Philippe SEKSIK, MD, PhD

(1)

Gastroenterology Unit Saint-Antoine Hospital, Paris

Set the scene for targeting the microbiome

ERL INSERM U1057 / UMR7203

(2)

• Dealing with complexity

• The tools

• Potential targets for gut microbiota manipulation

(3)

• The tools

(4)

Bacteria

Milieu

Virus

(Phages) Bacteriocines Quorum sensing

Nutrients AcidesBile

Xenobiotic

(5)

Bacteria

Milieu

Virus

Nutrients AcidsBile

Xenobiotic

(6)

Bacteria

Milieu

Virus

Xenobiotic

(7)

Bacteria

Milieu

Virus

Xenobiotic

Dynamics

Permanent instability

(8)

Theory of the ball in a cup

Dynamics

Permanent instability

(9)

RESILIENCE

(10)

• Complexity of the system

• Dynamics

Dealing with complexity

(11)

• Dynamics

• Complexity of the ‘diet’

• Dynamics

Dealing with complexity

(12)

• Dynamics

• Complexity of the ‘diet’

• Dynamics

Multi-scale Complexity

Dealing with complexity

(13)

Implementation & evolution

(14)

Delivery

Khoruts et al. Nature Medicine 2016 Backhed et.al Cell Host &Microbes 2015

Implementation & evolution

(15)

Delivery

Khoruts et al. Nature Medicine 2016 Backhed et.al Cell Host &Microbes 2015 Fallani et al. JPGN 2010 Breastfeeding /

infant formula Diversification

Implementation & evolution

(16)

Delivery

Khoruts et al. Nature Medicine 2016 Backhed et.al Cell Host &Microbes 2015 Fallani et al. JPGN 2010 Jackson et al. Gut 2016 Breastfeeding /

Drugs, Antibiotics Food

Implementation & evolution

(17)

Delivery

Khoruts et al. Nature Medicine 2016 Backhed et.al Cell Host &Microbes 2015 Fallani et al. JPGN 2010 Jackson et al. Gut 2016 Breastfeeding /

Drugs, Antibiotics Food

MICROBIOME SETTLEMENT

Implementation & evolution

(18)

Delivery

Khoruts et al. Nature Medicine 2016 Backhed et.al Cell Host &Microbes 2015 Fallani et al. JPGN 2010 Jackson et al. Gut 2016 Koren et al. Cell 2012 Breastfeeding /

Drugs, Antibiotics

Food Pregnancy

Drugs, Antibiotics

Food

MICROBIOME SETTLEMENT

Implementation & evolution

(19)

• The tools

(20)

Microbiota 16S analysis

Tissu

sample Conservation

-80°c

Critical steps

(21)

Tissu

-80°c

16S rRNA genes amplicons Mechanical and

chemical lysis

DNA extraction

Critical steps

(22)

Tissu

-80°c

chemical lysis

qPCR

Log10CFU/g

* *

7,00 7,50 8,00 8,50 9,00 9,50 10,00 10,50 11,00 11,50 12,00

Bacteria Bacteroides Firmicutes F. prausnitzii

Critical steps

(23)

Tissu

-80°c

chemical lysis

sequencing qPCR

Log10CFU/g

* *

7,00 7,50 8,00 8,50 9,00 9,50 10,00 10,50 11,00 11,50 12,00

Filtering

Critical steps

(24)

Tissu

-80°c

chemical lysis

sequencing qPCR

Log10CFU/g

* *

7,00 7,50 8,00 8,50 9,00 9,50 10,00 10,50 11,00 11,50 12,00

Sequences assignment to database or to OTU (97% similarity)

Diversity (alpha) : abundance within a sample

Diversity (beta) : PCA

Filtering

Critical steps

(25)

R package = pipeline for microbiota analysis

Extraction of information

1

Statistics 4

Diversity 5

Quality control

2 3

Data consolidation

6 specificity

Data processing

Data analysis

http://www.R-project.org

Harmonization Clean data

(26)

Description

• 16SrRNA Genes  composition in a complex ecosystem

 Which microbes are there

(27)

Toward functions

• 16SrRNA Genes  composition in a complex ecosystem

 Which microbes are there

• Need : to know the functions

 meta-Omics

(28)

Human gut microbiota

Abundance : 10¹⁴ bacteria Diversity : ~ 1000 species

Our other genome

(29)

Sum of all bacterial DNAs = Metagenome

Human gut microbiota

Our other genome

(30)

Metagenome (bacterial DNA): ~10⁸genes 100 to 150 x the human genome

Sum of all bacterial DNAs = Metagenome

Human gut microbiota

Our other genome

(31)

Mullard A. The inside story, Nature 2008

‘Metagenomic’ Projects

(32)

Microbiome analysis

sample

Metagenomic

DNA

Shot-gun sequencing

(33)

Microbiome analysis

sample Metatranscriptomic

Metagenomic

DNA

RNAs

(34)

Microbiome analysis

sample Metatranscriptomic

Metagenomic

Proteomic

Metabolomic

DNA

RNAs

Proteins

metabolites

(35)

R package = pipeline for microbiota analysis

Extraction of information

1

Statistics 4

Diversity 5

Quality control

2 3

Data consolidation

6 specificity

Data processing

Data analysis

http://www.R-project.org

Harmonization Clean data

(36)

Human gut microbiota

Highly complex : 10¹⁴ bacteria Highly Diverse : ~ 1000 species

Implementation from birth to 2 yrs-old

‘relative stability’  resiliency

A real organ with key functions

(37)

Bacteria Fungi

sIgA

Epithelium

IgA Tight

Jonctiuns Paneth cells

M cells Milieu

Mucus

Virus

anti-microbial peptides Bacteriocines

Quorum sensing

Nutrients Bile acids

Xenobiotiques

Treg Population

Treg/Th17 Adaptive and innate immunity

(38)

sIgA

Epithelium

IgA Tight

Mucus

Virus

Quorum sensing

Xenobiotiques

Treg Population

Metabolic pathways Fat storage

Glucose regulation

(39)

sIgA

Epithelium

IgA Tight

Mucus

Virus

Quorum sensing

Xenobiotiques

Treg Population

Metabolic pathways Fat storage

Glucose regulation Behavior

Gut  Brain

(40)

• The tools

(41)

Dysbiosis

Imbalance within symbiotic function between microbiome and host

(42)

Bacteria

Milieu

Virus

(43)

Bacteria

Milieu

Virus Direct Action on host

(44)

Bacteria

Milieu

Virus

(45)

Bacteria

Milieu

Virus

(46)

Bacteria

Milieu

Virus Diet mediated-Action on host CO-METABOLISM

(47)

Targets

• Phages

• Biles acids

• Tryptophane derived-metabolism by gut microbiota  ERC starting Grant 1,5M€

• Quorum sensing molecules

• Diet

(48)

Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut

Reyes A et al. PNAS Dec 2013; 110(50): 20236–20241

(49)

Gnotobiotic mouse model of phage–bacterial host dynamics in the human gut

Reyes A et al. PNAS Dec 2013; 110(50): 20236–20241

(50)

Biles acids

Duboc et al. GUT 2013

(51)

Biles acids

Duboc et al. GUT 2013

(52)

LuxI AHL

Quorum sensing mediated by AHLs

Bacteria Changes in phenotypes & in bacterial populations

(53)

LuxI LuxR

AHL

Lux box

Quorum sensing mediated by AHLs

Bacteria Changes in phenotypes & in bacterial populations

(54)

3oxo C12 AHL

Wild type mice

AHL in Gastric Tube

No AHL

1mg/Kg AHL 5mg/Kg AHL 10mg/Kg AHL

AHL 3 oxoC12 in gut microbiota

(55)

Impact of quorum sensing molecule (3 oxoC12) on mice gut microbiota

D14-21

(56)

Conclusions

• Microbiome : a new player in physiology

• Microbiome manipulation : therapeutic option

• Microbiome manipulation

 Targets

Phages, biles acids, Tryptophan-derived metabolism, Quorum Sensing molecules

 Diet = potential target

(57)

Conclusions

• Facing complexity driven by medical science

 We need

• Data managment / data analysis

• Dialog clinicians, scientists, ‘dry’ researchers

• Attract specialists in algorythms / non-linear statistics into microbiome science