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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