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Host-Pathogen Interactions in Tuberculosis

Olivier Neyrolles

Institute of Pharmacology & Structural Biology - CNRSToulouse - France

>1.5 million deaths15 million TB cases

2 billion latently infected individuals (?)

Only 5-10% exposed individuals develop TB

• Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)Science 2006

PLoS Med 2007

Am J Respir Crit Care Med 2007

Multiple factors for a complex disease

• Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)

TB / HIV

Multiple factors for a complex disease

Source: WHO 2005

• Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)

Multiple factors for a complex disease

Source: WHO 2005

Brazil • Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)

Multiple factors for a complex disease

• Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)

PLoS Med 2006

Lancet 2000

Am J Hum Genet 2000

Multiple factors for a complex disease

Filliol et al. 2006 J Bacteriol

W-Beijing lineage

• Nutritional status & hygiene

• HIV co-infection

• Sex • Age • Host genetic factors

• Infecting strain (virulence)

Multiple factors for a complex disease

19521957

196219212007

Public health measures

New vaccine

New drugs

WHO projections by 2020

• 1 billion people newly infected

• 200 million people sick

• 35 million deaths

M DC

CCR2

CRs, TLRsSRs, CLRs

IL12

CD8

CD4

MHC I

MHC II

CD1

CCR7

Lung

Lymph node

IL7/IL15

Effectors

Memory cells

CD4

CD8

IFNPerforin

Granulysin

IL23IL17

TNF

TregIL10

TGF

From D. Russell

M. tuberculosis cell entry

M. tuberculosis intracellular survival

Host cell response to infection

Fc receptor Complement

receptors (CRs)Mannose

receptor (MR)

DC-SIGN

Surfactant protein A/D

receptor (SPA/DR)

Scavenger

receptors (SRs)

ComplementImmunoglobulin Mannose residues

Surfactant proteins A/D

Lysosomes

Armstrong & Hart 1971 J Exp Med

Schlesinger 1993 J Immunol

Hirsch et al. 1994 J Immunol

Schlesinger et al. 1994 J Immunol

Schorey et al. 1997 Science

Tailleux et al. 2003 J Exp Med

Geijtenbeek et al. 2003 J Exp Med

Tailleux et al. 2005 PLoS Med

Gaynor et al. 1995 J Immunol

Beharka et al. 2002 J immunol

Zimmerli et al. 1996 Am J Respir Cell Mol Biol

Philips et al. 2005 Science

From Figdor et al. 2002 Nat Immunol

DC-SIGN & other phagocyte C-type lectins

High mannose

1,3-glucan

Man, GlcNAc,

Fuc, s6SLeX

Mannan, high-

mannose,

ManLAM , Fuc,

LeX, LeA, LeY,

LeB, 6SLeA

Man, Fuc, sLeX

Carbohydrates

IL4

IL10 IL2 IL6

IL23 IFNs,

ROS

TLR-dep TNF

TLR-dep IL12

?

TLR-dep IL12

TLR-dep IL10

IL10

TLR-dep IL12

Response

??Mo, B, CD4+ TC. albicansLigand on

CD4+CD25+ T cellsDectin-2

++DC, LC, M ,

PMN

P. carinii, C. albicans,

A. fumigatus

Ligand on T cellsDectin-1

??LC, DC subsetsHIV, M. lepraeType I procollagenLangerin

++DC, M subsetsHIV, HCV, CMV,

filoviruses, dengue, H.

pylori, M. tuberculosis, M.

leprae, S. mansoni, C.

albicans, A. fumigatus,

Leishmania spp.

ICAM-2/3,

CEACAM-1, Mac-1,

CEA

DC-SIGN

?+DC, LC, Mo, MHIV, P. carinii,

M. tuberculosis,

C. albicans

Lysosomal

hydrolases,

L-selectin, MUC-1

MR

SignalingEndocytosisCell typesPathogen bindingEndog. Ligand(s)

DC-SIGN & other phagocyte C-type lectins

DC-SIGN is a major M. tuberculosis receptor in human DCs%

Bin

din

g t

o h

DC

s

Blocking antibodies

DC-SIGN M. tuberculosis Merged

Tailleux et al. 2003 J Exp Med

DC-SIGN is a major M. tuberculosis receptor in alveolar M s in TB patients

Tailleux et al. 2005 PLoS Med

DC-SIGNM. tuberculosis Merged

DC-SIGN is a major M. tuberculosis receptor in alveolar M s in TB patients

DC-SIGN recognizes mannosylated ligands in the M. tuberculosis envelope

Maeda et al. 2003 J Biol Chem

Pitarque et al. 2005 Biochem J

Differential mycobacterial behavior?

Pro / anti-inflammatory pathway?

Protection / susceptibility?

Primo-infection Established Infection

Complement receptors (CRs)

Mannose receptor (MR) DC-SIGN ( / ManLAM…) >> CRs - MR

M. tuberculosis cell entry

M. tuberculosis intracellular survival

Host cell response to infection

M DC

CCR2

CRs, TLRsSRs, CLRs

IL12

CD8

CD4

MHC I

MHC II

CD1

CCR7

Lung

Lymph node

IL7/IL15

Effectors

Memory cells

CD4

CD8

IFNPerforin

Granulysin

IL23IL17

TNF

TregIL10

TGF

TLR1/6TLR2 TLR4

CD14MD2

DC-SIGN

Gene transcription

Mycobacterial ligands

Geijtenbeek et al. 2003 J Exp Med

Gringhuis et al. 2007 Immunity

NF- B

Raf-1

p50

p65 (RelA)

Acetylation

Signaling through DC-SIGN might influence host cell response to infection

TLR1/6TLR2

Dectin

Gene transcription

Mycobacterial ligands

Rothfuchs et al. 2007 J Immunol

NF- B

Syk

Other C-type lectins influence host cell response to infection

TLR4

CD14MD2

Dectin blockade

M. tuberculosis cell entry

M. tuberculosis intracellular survival

Host cell response to infection

From D. Russell

From Cole et al. 1998 Nature

« This history, together with the clues to conquer the tubercle bacillus, is written in its genome.

(…) we now have the sequence of every potential drug target and of every antigen we may

wish to include in a vaccine …»

« … But can we convert this mass of information into a usefull understanding? »

Douglas B. Young 1998

Function unknown 41%

Genome comparison Mutant library screening Transcriptomics & proteomics

Gene candidate

High throughput drug screeningKnowledge-based approach

Experimental genetics & functional genomics can make the information more understandable

Camacho et al. 1999 Mol Microbiol

Cox et al. 1999 Nature

Camacho et al. 2001 J Biol Chem

Signature-tagged Transposon Mutagenesis (STM) has allowedthe identification of the first virulence genes cluster in M. tuberculosis

hM

M. tuberculosis (STM library)

2 x 7-8 days

PCR / Hybridisation

Candidates

Individual confirmation

LM-PCR / Sequencing

Identification of M. tuberculosis genes involved in M parasitism by STM

Signature-tagged Transposon Mutagenesis

Mutant library

n=48

Pooled mutants (input)

Selection

(mice, macrophages…)

Output

Genomic DNA

Real time-PCR-amplified tags

Signature-tagged Transposon Mutagenesis

(STM)

Rosas-Magallanes et al. 2007 Infect Immun

Rosas-Magallanes et al. 2006 Mol Biol Evol

M. tuberculosis genes involved in M parasitism

Disrupted gene Putative function

mmp L 2 Fatty acid transport ?

Rv0097 Oxidoreductase ?

dr rB PDIM transport

Rv2954c/5c PDIM/PGL synthesis ?

Rv2958c PDIM/PGL synthes i s

a lkB Fatty acid metabolism

papA1 Sulfolipid synthesi s

pks6 Polyketide synthase

Rv0986 Cell adhesi o n

yrbE4A Cell adhesi o n

bfrB Iron storage

Rv1817 Flavoprotein ?

moaC1 Molybdopterin biosynthesis ?

moaX Molybdopterin biosynthesis ?

Rv2104c/5c Toxin/Antitoxin

Rv2336 Unknown

ppe5 Unknown

ppe8 Unknown

Rv1502 Unknown

Rv2227 Unknown

Lipid metabolism & cell wall biogenesis

Bacterial metabolism (iron storage & alternative respiration)

Proteins with unknown function

Mtb / DAMP

pH~6.3

vATPase

pH~5.0-5.5

vATPaseKilled Mtb / DAMP

« Is the nonfusion response to M. tuberculosis due to the

surface properties of the bacterium or to an active inhibitor

derived therefrom? Does it represent a fixed character or

can manipulation cause a fusion response to be

substituted? If so, what is the effect of such conversion on

subsequent intracellular bacterial survival and

multiplication?»

M. tuberculosis intracellular survival & trafficking

The latex bead phagosome (M. Desjardins)

The mycobacterial phagosome

Early endosomes Recycling endosomes

***

*

* *

**

Transferrin

Rab5

pH 6.5

Lysosomes

vATPase

Armstrong & Hart 1971 J Exp Med

Sturgill-Koszycki et al. 1994 Science

Clemens & Horwitz 1995 J Exp Med

De Chastellier et al. 1995 Eur J Cell Biol

Sturgill-Koszycki et al. 1996 EMBO J

Clemens & Horwitz 1996 J Exp Med

Schaible et al. 1998 J Immunol

Via et al. 1998 J Cell Sci

The mycobacterial phagosome

From C. de Chastellier

SapMPknG

LAM

PI3P PI

p38MAPK

VPS34GDI

GDP

Rab5-GDP

EEA1-PI3P/Stx6-

mediated V0H+ ATPase &

lysosomal enzyme delivery

The M. tuberculosis phagosome

PI3PPI

De Chastellier & Thilo 1997 Eur J Cell Biol

Walburger et al. 2004 Science

Vergne et al. 2005 PNAS

Vergne et al. 2003 J Exp Med

Fratti et al. 2003 J Biol Chem

Cavalli et al. 2001 Mol Cell

Fratti et al. 2003 PNAS

LAMP1 (-WT)

LAMP1 (+WT)

PI3P is necessary for phagosome maturation

Vieira et al. 2001 J Cell Biol

Mycobacteria secrete a PI3P hydrolyzing phosphatase SapM

Vergne et al. 2005 PNAS

Macrophages

Iron-dextran

Mutant pools

Infected macrophages Cell lysis

& magnetic sorting

Phago-lysosomes

Phagosomes

Sequencing

Mutant pools

Infected macrophages

Lysotracker staining

Cell lysis

& flow sorting

Phago-lysosomes

Phagosomes

Sequencing

Lysosomes

Macrophages

Lysosomes

Magnetic sorting-based screening at the phagosome level

Pethe et al. 2004 PNAS

Fluorescent sorting-based screening at the phagosome level

Stewart et al. 2005 PLoS Pathog

M. tuberculosis genes involved in M parasitism

Disrupted gene Putative function

mmpL12 Fatty acid transport ?

fadD26 PDIM/PGL synthes i s

fadD28 PDIM/PGL synthes i s

l i pO Fatty acid metabolism ?

papA1 Sulfolipid synthesi s

pks6 Polyketide synthase

Rv1819c ABC transporter Rv0986 Cell adhesi o n

Rv0249c Succinate dehydrogenase ?

nuoL NADH dehydrogenase ?

Rv2336 Unknown

pe22 Unknown

ppe19 Unknown

Rv3527 Unknown

Rv0918 Unknown

Rv3378c Unknown

Lipid metabolism & cell wall biogenesis

Bacterial metabolism (respiration)

Proteins with unknown function

Pethe et al. 2004 PNAS

Institut Pasteur, Paris

Unit of Mycobacterial Genetics

Brigitte Gicquel

Ludovic Tailleux

Mary Jackson

Nathalie Winter

Norihiro Maeda

Vania Rosas Magallanes

Yann Bordat

Frédéric Boudou

Jean Rauzier

Patricia Charles

National collaborators

Institut Pasteur, Paris

Lluis Quintana-Murci

Olivier Schwartz

Roland Brosch

INSERM

Jennifer Becq (Paris)

Patrick Deschavanne (Paris)

AP-HP, Paris

Philippe Lagrange

Jean-Louis Herrmann

Pierre Scheinmann

Jacques De Blic

Abdelatif Tazi

Paul Fornès

International collaborators

Graham Stewart, University of Surrey, UK

Douglas Young, Imperial College London, UK

Kurt Drickamer, Imperial College London, UK

Paola Castagnoli, University of Milan, Italy

Priscille Brodin, IP Seoul, Korea

Qian Gao, Fudan University Shanghai, China

IPBS - CNRS, Toulouse

Antoine Tanne, PhD student

Yannick Poquet, Ass. Prof

Florence Levillain, Technician

Pascale Salek, Post-doc

Hélène Botella, MSc

Alexandre Gouzy, MSc

Jérome Nigou

Germain Puzo

Team, collaborations and funding