CEA CHRU CNRS CPU INRA INRIA INSERM INSTITUT PASTEUR IRD

From bugs and men to treatments and vaccines

Philippe Sansonetti & collaborators

Chaire de Microbiologie et Maladies Infectieuses,Collège de FranceUnité de Pathogénie Microbienne Moléculaire,& Unité INSERM 786Institut Pasteur

3èmes rencontres internationales de la rechercheJune 10, 2011

Symbionts

Innate immunityPhysiological inflammationSurveillance/Tolerance

Recognition network:TLRs,NLRs,

Rig1, MDA5…Danger signals:

(uric acid, ATP, cytochrome C,etc..)

Pathogens

Innate immunityPathological inflammationMicrobe & tissue destruction

Amplification loop:TREM, HMGB1, Gal3,

Severe sepsisSeptic shock

Regulation

Loss of control

Adaptive immunityPathogens recognition,capture, completion of

eradication process, protection

Sansonetti, 2004, Nature Rev. Immunol.Sansonetti, 2006, Nat. Immunol.Sansonetti & Di Santo, 2007, ImmunitySansonetti & Medzhitov, 2009, CellSansonetti, 2010, Mucosal Immunology

Man is a primate-microbes hybrid

Concept of HOLOGENOME / SUPERORGANISM

- Rupture of homeostasis = IBD- Dysbiosis = obesity, diabetes,metabolic syndromeTurnbaugh & Gordon. 2009. J.Physiol.

Human colon: 1011 bacteria / g feces500-1000 species,10x human cells150x human genes Quin, 2010, Nature

Bacterial life at mucosal surfaces« Seating on a volcano »

O2NOROSAnti-microbial peptidesOther: lysozyme, proteases, lectins, phospholipasesTransmigrating phagocytes

Epithelium

Mucus

Cationic antimicrobial peptide hBD3

SurviveSubvert

Arbibe et al., Nature Immunol., 2007Sperandio et al., J. Exp. Med., 2008Marteyn et al., Nature, 2010

Pro-inflammatorycascade

TLRTLR

NLRNLR

Pro-inflammatory genesPro-inflammatory genes

PMNPMNActivated DC & MActivated DC & MΦΦ Th1 - Th17 / Th1 - Th17 / TregTreg

OCTN2OCTN2 PepT1PepT1

QSM QSM MDPMDP

TLRTLR

NLRNLRRegulatorycascade

??

Regulatory genesRegulatory genes

MucusMucus

Regulatory cytokines, chemokinesRegulatory cytokines, chemokines

Immature DC & MImmature DC & MΦΦ Treg / Treg / Th1 – Th17Th1 – Th17

PATHOGENS MucinasesAdhesins / InvasinsType III / IV secretory systemsHemolysinsMassive engagement of TLRs, NLRsEradication of commensal microbiota (niche occupancy)+ Dampening innate / inflammatory responses

SYMBIONTSAbsence (limitation) of virulence factorsPAMPs less agonist ?Sequestration, weak activity of TLRs Life in biofilms on mucus surfaceControled diffusion and sampling of PAMPsand prokaryotic signalisation molecules, includingregulatory molecules (i.e. PSA of B. fragilis (Mazmanian & al.)

AntimicrobialAntimicrobialmoleculesmolecules

Pro-inflammatory cytokines, chemokines Pro-inflammatory cytokines, chemokines

PMNPMNDC

Phy

siol

ogic

al in

flam

mat

ion

Phy

siol

ogic

al in

flam

mat

ion T

olerancebT

oleranceb

Pat

holo

gica

l inf

lam

mat

ion

Pat

holo

gica

l inf

lam

mat

ion

PATHOBIONTSSFBH. hepaticus

Effectors ?

Intestinal lumen « Milieu intérieur »/ organs

: PAMPs,other bacterialfactors/effectorsmetabolites

CNS (maturation, behaviour,

inflammation)

Vessels (maturation,

inflammation)

Adipose tissue(insulin resistance, obesity,

diabetes)

Liver (sugar & lipid metabolism)

Lymphoid organs(maturation, mucosal & systemic

physiological inflammation)

Microbiotaas an extra organ

PAMPs, as hormones + mediators produced by the epitheliumunder « pressure » of the microbiota

- Crossing of organ barriers(endothelium, BBB, etc…)- Receptors / specialized cells

: Host mediatorschemokinescytokineshormonesneuromediators

Intestinal epithelial barrier

The host-microbiota symbiosis affects development, metabolism, immunity, what else ?

Signature Tagged Mutagenesis in Lactobacillus caseiHélène Licandro, Jean-Françios Cavin, Thierry Pédron

Sequencing of mutations

Assembling in pools of high interest

“Cell surface”

“Regulation”

“Random”

GavageQuantification

of mutantsTransposase production

pVI110 transposition

X72

Development of an efficient tool to generate random mutants of L. casei with a two-step procedure.

Adaptation for STM and generation of a 7000 random tagged-mutants library.

All mutations currently sequenced (Sanger Center) to optimize the use of STM by assembling tagged mutants in pools of predicted high physiological interest.

Tagged mutants pools tested in a mouse model to determine genes involved in colonization and understanding of the role played by these genes.

In vitro/in vivo assays to identify effector functions on cell cycle, cell differentiation, metabolism, anti-inflammation, etc…

ColonFollicle-associated

epithelium

Small intestine

Mucus

Pro

lifer

ativ

e c

ompa

rtm

ent

(PC

)D

iffer

entia

ting

com

part

men

t (D

C)

Desquamatingcells

Cell cyclearrest

Stem cells (SC) compartment Stem cells compartment

Cell cyclearrest

PC

DC

Paneth cell Crypt-Based Columnar (Lgr5+) SC

+4 Radiation-resistant SC Crypt-Based Columnar (Lgr5+) SC Paneth cells ?

M cellsDendritic cellsMacrophages

T lymphocytes

B Lymphocytes

IEC

Panet

h ce

ll

Goblet

cell

Enter

ochr

omaf

fin ce

ll

The 4 gut epithelial lineagesPro

lifer

ativ

epr

ogen

itors

Abs

orbt

ive

secr

eto

ry c

ells

Lieberkühn’scrypt

A crypt-specific core microbiota in gut homeostasis, restitution… and cancer ?

Crypt stem cells (Lgr5+)

Lgr5-GFP

Crypt-specific core microbiota

FISH

Epithelial cells

mucus

Basolateral macropinocytosis (TTSS)

Vacuole lysis(TTSS)

Escape to autophagy

Motility, cell to cellspread (TTSS)IcsA

??

M cell

MΦ

B Lympho

MΦpyroptosis

TTSS/IpaB

-Pyroptosis = proinflammatory apoptosis-Activation of caspase-1, Release of IL-1β and IL-18

DC

«facilitated translocation»

Follicle-associated epithelium

PNN

Nod1

PGN

NF-κBJNK

TTSS

Pro-inflammatorygenes

IL-8, other cytokineschemokines

- Development of inflammation- Rupture of epithelial barrier- Facilitation of invasion- Stimulation of epithelial bactericidal capacities

Defensins and other bactericidalmolecules

CCL-20

Rupture, invasion and inflammatory destruction of the intestinalepithelium by Shigella: the key steps of TTSS function,

a gold mine to identify new effectors and pathways of innate immune responses

B Lympho

T Lympho

VirB

ipaA, ipaB, ipaC, ipaD,ipgB1, ipgD, icsB,ospC2/3/4,ospD1, ospD2

ospD3, ospE1/2, ospG,ipaH1/2, ipaH4,ipaH7, ipaH9.8

MxiE

Expression / regulation / function of type III effectors

before secretion after TTSS activation(target cell recognition)

INVASION Modulation of INNATE RESPONSES

IpaB, IpaC, IpaA, IpgB1,VirA, IpgD

IpgD: phosphatidyl-inositol phosphatase, hydrolyses P in 4 in Pi(4,5)P2 (Niebuhr et al, 2002, Pendaries et al, 2006 EMBO J.). Anti-inflammatory +++ (Puhar et al., in preparation).

OspG: kinase,binds/blocks ubiquitin transfer protein E2, protects I-kB from degradation. Anti-inflammatory +++ (Kim et al., 2005, PNAS).

OspF: dephosphorylation of Erk1/2, epigenetic regulation of pro-inflammatory genes - i.e. IL-8. Regulates transmigrationof PMNs through epithelium (Arbibe et al., 2007,Nat.Immunol.). Phosphothreonine lyase (Li et al., 2007, Science).

IpaHs: (5 + 5 chromosomal copies): New family of Ubiquitin ligases (E3) (Rohde et al., 2007, Cell Host & Microbes)IpaH9.8 targets NEMO (Ashida et al., 2010, Nat.Cell Biol.)

OTHER PHENOTYPES

IcsB: inhibion or autophagy(Ogawa et al., 2005, Science)

VirA: inhibition of microtubules, facilitates actin-based motility(Yoshida et al., 2006, Science)

ospBospFospC1virA

cellules épithéliales

mucus

macropinocytosebaso-latérale (TTSS)

lyse vacuole(TTSS)

motilité/passagecellule-cellule

(TTSS)IcsAM cell

MΦ

Lympho B

MΦpyroptosis

TTSS/IpaB

- activation of caspase-1-pyroptosis = pro-inflammatory apoptosis- release of IL-1β et IL-18 DC

«facilitated translocation»

follicle-associated epithelium

Nod1

PGN

NF-κBJNK

TTSS

Osp(s)TTSS

IL-8

CCL-20

PNN

Suppression of humoral defense mechanismsSuppression of humoral defense mechanisms

Suppression of cellular defense mechanismsSuppression of cellular defense mechanisms

AMPs

Arbibe et al. Nat. Immunol., 2007Sperandio et al., J. Exp. Med., 2008Puhar et al., in preparation

ATP

ATP

Suppression of danger signaling

PR

Tran Van Nhieu et al., Nat. Cell Biol. 2003Puhar et al., in preparation

ATP = danger signal

Inflammasome activationDifferentiation of naive T cells to « inflammatory » Th17 cells

HEMICHANNEL(Connexins) x

dxd

xd

xd

xd

xd

xd

xd

xd

IpgD

Pi(4,5)P2 Pi(5)PIpgD

IpgD neg. phenotype wt phenotype

Pi(5)P

IpgD impairs danger signaling

IpgD impairs T cell polarization

PIP2 binding causes ERM conformation change between inactive and active / phosphorylated forms

ERM proteins Ezrin, Radixin, Moesin: crucial role in cell polarization during T lymphocyte migration

Lee JH et al. 2003

IpgD-mediated PIP2 cleavage with subsequent reduction of PIP2 pool at the plasma membrane.What about pERM ?

IpgD injection/expression causes hydrolysisof Pi(4,5)P2 and dephosphorylation of Ezrin

IpgD-mediated hydrolysis of PI(4,5)P2causes activated T cell depolarizationand loss of oriented movement in presence of chemokine CXCL12Konradt et al., Cell Host Microbe, 2011

CD4+ T cell dynamics at 4hm

m/m

in

Velocity

Uninfected T3SS- WT

Uninfected T3SS- WT

Str

aigh

tnes

s

Confinement

Uninfected T3SS- WT

****** ***

%

Arr

est

Arrest

Uninfected T3SS- WT

******

Salgado-Pabon et al., in preparation

PerspectivesTo continue to decipher the pathways of innate and adaptive protection against pathogens by analysing how bacterial « anti-immunity » effectors subvert the molecular and cellular mechanisms of immune defenses (i.e. target identification)

To identify the molecular effectors and mechanisms by which symbionts regulate host local and systemic innate and adaptive immune responses, and other key areas reflecting the duration, intimacy and depth of the host-microbe symbiosis (i.e.: metabolism, tissue repair, cancer…)

To rationally develop novel therapeutics and vaccines based on understanding microbe-host cross talks