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The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
1The screen versions of these slides have full details of copyright and acknowledgements
1
The Genetic Structureof Mycobacterium tuberculosis
Insights into Genome Organisation and Evolution of the Causative Agent of Tuberculosis
Roland Brosch Ph.D.Integrated Mycobacterial Pathogenomics Unit
Institut Pasteur Paris, France
2
Bacteria
Protobacteria Cyanobacteria FirmicutesActinobacteria
Fast growing mycobacteria Slow growing mycobacteria
Nocardia CorynebacteriumMycobacteriumetc.
http://wwwabi.snv.jussieu.fr/~erocha/research/ordervsdisorder.html
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M. leprae
M. avium paratb.
M. marinum
M. tuberculosis complex
M. ulcerans
16S rRNA tree -Springer et al., J. Clin. Microbiol., 1996
Slowly growing mycobacteria
• Etiological agent of buruli ulcer• Aquatic mycobacterium• Produces mycolactone
• Etiological agent of leprosy• Reductive evolution
• Fish pathog. & human opportun. pathogen• Aquatic mycobacterium
Johne’s disease
• Etiological agent of human tuberculosis (TB)
• 8-10 million new cases• 1.7 million deaths / year• 2 billion infected
• M. canettii• M. tuberculosis• M. africanum• M. orygis• M. microti• M. pinnipedii• M. bovis
— BCG
Selected slowly growing mycobacteria
etc.
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
2The screen versions of these slides have full details of copyright and acknowledgements
4
Question: What are the genetic determinants for the evolutionary success of M. tuberculosis?
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4,4 Mb
Genome of M. tuberculosis H37RvFirst mycobacterial genome sequenced
• Genes for aerobic, micro-aerophilic and anaerobic growth
• Abundance of genes involved in lipid metabolism
• Eukaryotic-like Serine/Threonine Protein Kinases
• Novel gene/protein families e.g., PE/PPE, ESAT-6
Cole ST, Brosch R, Parkhill J, et al., 1998, Nature 393: 537-544
GC content 65.6%
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M. tuberculosis H37Rv: ~ 4000 genes annotated
Cole et al., Nature 1998
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
3The screen versions of these slides have full details of copyright and acknowledgements
7
M. tuberculosis H37Rv: zoom-in
8
Broad classification of M. tuberculosis genes
Class FunctionNumber of genes
Total length (kb)
% total coding
0. Virulence, detoxification, adaptation
1. Lipid metabolism
2. Information pathways
3. Cell wall and cell processes
4. Stable RNAs
5. Insertion sequences and phages
6. PE and PPE proteins
7. Intermediary metabolism and respiration
8. Proteins of unknown function
9. Regulatory proteins
10. Conserved hypothetical proteins
Potentially non-coding sequences
91
225
207
517
50
137
167
877
607
188
911
95
372
243
620
10
100
283
985
396
162
739
434
2.4
9.3
6.1
15.5
0.2
2.5
7.1
24.6
9.9
4.0
18.4
Cole et al., Nature, 1998
9Sassetti et al., Mol. Microbiol 2003
The power of high density transposon mutation screens:
Alternative method: postgenomic prediction using Bayesian statistical analysis (Markov chain Monte Carlo)Lamichhane et al., PNAS, 2003
Transducing Phage phiMycoMarT7 Himar1 mariner transposon
~100 000 mutants
How to identify the essential genesof M. tuberculosis ?
Sassetti et al., PNAS, 2001
Wild-Type Transposon mutant library
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
4The screen versions of these slides have full details of copyright and acknowledgements
10Sassetti et al., Mol. Microbiol, 2003; Sassetti & Rubin, PNAS, 2003
Transposon site hybridization (TraSH)predicts ~ 600 essential genes (in vitro)
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High density transposon mutation screens & Next Generation Sequencing (NGS)
Griffin et al., PLoS Pathog, 2011
12 Griffin et al., PLoS Pathog, 2011
Example for an essential gene: pknB ... encoding the serine/threonine protein kinase PknB
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
5The screen versions of these slides have full details of copyright and acknowledgements
13
M. tub. Serine Threonine Protein Kinases (STPK)
Kinase domain
Transmembranedomain
b-propeller
Pro-rich
Trx motif TPR motif
Regulatory region
PASTA domains
PknG
PknK
PknA
PknE
PknF
PknI
PknJ
PknL
PknD
PknH
PknB
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Genomic region pknB - a conserved operon
Fernandez et al., J. Bact. 2006
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pknB is essential for M. tuberculosis
Similar results were obtained for pknB of M. smegmatis
WT
Cassette-insertion-pknB locus
Cassette insertion-integrating vector locus
Fernandez et al., J. Bact. 2006
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
6The screen versions of these slides have full details of copyright and acknowledgements
16
• erp (Rv3810)Berthet, F.-X. et al., Science, 1998
• phoP (Rv0757)Perez E. et al., Mol. Microbiol, 2001
• leuCHondalus et al., Infect Immun, 2000Bardarov et al., PNAS, 1997
Advances in mycobacterial genetics –identification of virulence factors
pAL5000 from M. fortuitumcounter selective marker(thermosensitive ori), sacB
Tools for gene inactivation
Thermosensitive phages
Mycoserosic acid synthasePhtiocerol and phenophtiocerol synthaseAcyl-CoA synthase
Transporter
STM (Signature Tagged Mutagenesis)
M. tuberculosischromosome
M. tuberculosischromosome
Cox JS, et al., Nature, 1999Defect in the synthesis of phtiocerol dimycoserosate (PDIM) involved in permeability of the cell envelope and its structure Camacho et al., Mol. Microbiol, 1999
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Genetic requirements for mycobacterial survival during infectionTraSH (Transposon site hybridization) using phage MycoMarT7 and micro-arrays
Sassetti & Rubin, PNAS, 2003
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Genes encoding the ESX-1 type VII secretion system: Example of genes predicted to be involved in virulence
Bitter et al., PLoS Pathog, 2009
Sassetti & Rubin, PNAS, 2003
Table 3. genes predicted to be required for in vivo survival
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
7The screen versions of these slides have full details of copyright and acknowledgements
19
Genome comparison
Alternative ways to predict genes involved in virulence:
M. tuberculosis H37Rv
Genetically very similar but attenuatedM. bovis BCG
M. microtiGenetically very similar but attenuated
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• ~ 3 x 109 doses used worldwide
• No reversion to virulent phenotype --> permanent genetic change e.g., deletion
• M. bovis BCG
Mycobacterium bovis BCG
230 passages
Calmette & Guérin
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1930: acid-fast bacillus causing a natural disease in field voles (Dr. Wells)
1950-1970: Vole bacillus used as live vaccine for the prevention of tuberculosis in the UK and in Prague
Mycobacterium microti
D’Arcy Hart & Sutherland, Br. Med. J.,1977; Sula & Radkovsky, J. Hyg. Epid. Microbiol. Immunol. 1976
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
8The screen versions of these slides have full details of copyright and acknowledgements
22
RD1BCG
ESAT-6 (6kD early secreted antigenic target)(EsxA)
RD1microti
Region of Difference 1 (RD1) oriC
BCG::RD1
attB
ΔRD1
esxA
PE/PPERv3869Rv3868
Rv3867
Rv3866
Rv3864/65
Rv3863
Rv3862
Rv3861 Rv3870 Rv3871 esxB
Rv3876
Rv3877 Rv3878
Rv3879
Rv3880
Rv3881
Rv3882
Rv3883
Rv3884 Rv3885
Rv3886
Rv3887
Rv3860
CFP-10 (10 kD culture(EsxB) filtrate protein)
• Found in culture filtrate of M. tub.
• No N-term signal sequence
• Triggers strong IFN-g response
• Used for TB diagnostics
Ab Anti-ESAT-6
(Kindly received from P. Andersen)
4354435343524351435043494348434743464345434443434342434143404339433843374336 4355 4356 4357 4358 4359 4360 4361 4362 4363 4364 4365 4366 4367 4368 4369 4370 4371 4372
Integrating cosmid pRD1-2F9 attP
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pH 7 pH 4 pH 7 pH 4- RD1 + RD1
Supernatant of in vitro liquid mycobacterial culture
CFP-10
The proteomes of RD1- and RD1+ tubercle bacilli differ for ESAT-6 and CFP-10
ESAT-6
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BCG::RD1 shows different colony morphology than the BCG pYub412-vector control
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
9The screen versions of these slides have full details of copyright and acknowledgements
25
pRD1-2F9 attP
ΔRD1BCG
4,350,266 4,359,717
oriC
BCGattB
ΔRD1
Cell lysate
Supernatant
Cell lysate
Supernatant
Ab a
nti-E
sxA
(ES
AT-6
)Ab
ant
i-Esx
B(C
FP10
)
pRD1-2F9
The RD1 region codes for a novel secretion system in mycobacteria: ESX-1
pe-ppe-esxB-esxAattP
pe-ppe-esxB-esxA
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oriC
M. microti
attB
ΔRD1
pRD1-2F9 attP
Simeone et al., Curr. Opin. Microbiol., 20094,340,417 4,354,536
ΔRD1mic
Cell lysate
Supernatant
Cell lysate
Supernatant
Ab a
nti-
Esx
A(E
SAT
-6)
Ab a
nti
Esx
B(C
FP-1
0)
pRD1-2F9
attPpe-ppe-esxB-esxA
pe-ppe-esxB-esxA
The RD1 region codes for a novel secretion system in mycobacteria: ESX-1 (2)
27 Bitter et al., 2009 PLoS Pathog.
Ecc = ESX conserved componentEsp = ESX-1 secretion-associated protein
Novel gene nomenclature for T7SS - ESX-1 serves as paradigm
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
10The screen versions of these slides have full details of copyright and acknowledgements
28
SCIDSevere CombinedImmuno-Deficient
Pym et al., Mol. Microbiol., 2002,
Re-integration of RD1 region into BCG reconstitutes the ESX-1 secretion system
and increases virulence
BCG::vector-ctrl
BCG::RD1-2F9
BCG::pYUB412
BCG::RD1-2F9
29Key element for host-pathogen interaction & vaccine and therapy strategies
• Phagosomal rupture & vacuolar escape
• Increased CD8 responses(Mtb ~ 200 fold stronger than BCG)
• NLRP3 inflammasome activation
• Type I interferon response
• Cell to cell spread
• Enhanced vaccine efficacy of ESX-1 vaccines
ESX-1 mediated processes differentiate BCG from M. tuberculosis for many cell-biological
and immunological aspects
Hsu et al., 2003, PNASGuinn et al., 2004, Mol. Microbol. Gao et al., 2004, Mol. Microbiol
Pym et al., 2003, Nat. Med.Brodin et al., 2004, J. Infect. Dis.
Stanley et al., 2007
Mishra et al., 2010, Cell. Microbiol. Dorhoi et al., 2012, Eur. J. Immunol.
Majlessi et al., 2005, J. Immunol.Billeskov et al., 2007, J. Immunol.Ryan et al., 2009, J. Immunol.
van der Wel et al., 2007, CellSimeone et al., 2012, PLoS Pathog.
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M. tuberculosis has 5 ESX/type VII systems Study of functions is underway
Bitter et al., 2009, PLoS Pathog.
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
11The screen versions of these slides have full details of copyright and acknowledgements
31
Comparison
Comparative genomics within the tubercle bacilli allows insight into the evolution of M. tuberculosis
M. tuberculosis H37Rv
M. bovis BCG
Other tubercle bacilli
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Host range of tubercle bacilli
Mycobacterium tuberculosis complex
CattleDeerGoats
HumansLions
LlamasSeals
VolesShrews
Field mice
Humans(mainly W-Africa)
Humans(everywhere)
M. tuberculosis M. africanum M. microti M. bovis
M. bovis BCGvaccine
Identical 16S rRNA gene sequence
99.9 % sequence similarity
M. canettiiHumans
(very rare)
SealsSea lions
M. pinnipedii
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Comparative genomics identifies Regions of Difference (RD)
Example of RD9RD region
RD flanking primers
RD flanking primersRD flanking primers
Brosch et al., PNAS, 2002
M. c
anet
tii
M. t
uber
culo
sis
M. a
frica
num
M. m
icro
ti
M. b
ovis
BC
G
M. p
inni
pedi
i
RD9flanking
kb
0.6
3.02.0
1.0
0.4
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
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34
M. africanum
M. microti
BCG
M. bovis
Sequence conservation of the RD9 junction region
in different subspeciesAAATTACTGTGGCCCACGCCGGGCCGG
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TbD flanking primers
TbD region
TbD flanking primers
TbD flanking primers
Comparative genomics identifies M. tuberculosis deletion region 1 (TbD1)
TbD1= M. tuberculosis deletion region 1
M. t
uber
culo
sis
M. c
anet
tii
M. a
frica
num
M. m
icro
ti
M. b
ovis
BC
G
M. p
inni
pedi
i
TbD1flanking
kb
3.02.0
1.00.60.4
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Region TbD1 absent from many M. tuberculosis strains
• M. bovis• M. africanum• M. microti• BCG• Ancestral M. tub.
• M. tuberculosis H37Rv • M. tuberculosis CDC1551• M. tuberculosis Beijing 210• M. tuberculosis C• M. tuberculosis F11
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
13The screen versions of these slides have full details of copyright and acknowledgements
37
Most junction regions of RDs are located within genes
Deletion events --> not insertion events
Deletion events and the M. tuberculosis complex
Conserved deletionsAmong members of the M. tuberculosis complex
Most probably occurred in a common progenitor strain
38 BCG Pasteur
Evolutionary pathway of the tubercle bacilli
Brosch et al., PNAS, 2002 Mostowy et al., J Infect Dis, 2002 Gutierrez et al., PLoS Pathog, 2005Smith et al., Nat Rev Microbiol. 2009Supply et al., Nat Genet. 2013
Common ancestor(s)
M. africanum
M. canettii and other smooth tubercle bacilli
M. microti
M. b
ovis
M. pinnipedii (seal)Oryx
Goat- M. caprae
“Classical”
BCG Tokyo
“Ancestral”, SE AsianTBD1-intact
BeijingCDC1551H37Rv
• 3 main lineages (“classic” MTBC)(TbD1+, ∆TbD1, ∆RD9) + Mcan
• Perfect correlation of RDs with SNPs (and resistanceto pyrazinamide)
• Mainly clonal population structure
• No recent horizontal gene transfer detectable
“Mod
ern”
TbD
1-de
lete
d
RDcanNumerous sequence polymorphisms
katGc463CTG→CGG
gyrAc95ACC→AGC
TbD 1
RD 7
RD 13
RD 1
RD 4
RD 2
RD 14
pncAc57CAC→GAC
oxyRn285G→ARD 12
RDseal
mmpL6551AAC→AAGRDmic
RD 9
RD 10RD 8
39 After Gagneux & Small., Lancet Infect Dis., 2007
TbD1-intact M. tuberculosis strains are predominant in South Asia
“Ancestral” TD1+ M. tub.
“Modern” ΔTD1 M. tub.
M. africanum
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
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40
The oldest case of human tuberculosis known in Britain (Iron age)
2,200 years ago
TbD1 deleted M. tuberculosis strain
41After Hershberg et al., PLoS Biol., 2008
Broad diversity of worldwide
M. tuberculosis strains
Multi-Locus-Sequence-Analysis (MLSA) based on 89 concatenated gene sequences
for 108 strains
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Filtered SNPs
After Comas et al., Nat. Genetics, 2010
Broad diversity of worldwide M. tuberculosis strains (genome data)
The Genetic Structure of Mycobacterium tuberculosis
Roland Brosch
15The screen versions of these slides have full details of copyright and acknowledgements
43
Conclusions and perspectives (Evolution of M. tuberculosis)
• The classical M. tuberculosis complex is a successful lineage that evolved mainly by clonal expansion from a pool of M. canettii-like tubercle bacilli
• Most genetic lineages of M. tuberculosis strains are geographically linked
• The branching of M. tuberculosis lineages seems to be thousands of years old
• M. bovis is not the ancestor of M. tuberculosis
• Information on genome content of different lineages allows now to experimentally address the factors that contributed to the evolutionary success of M. tuberculosis and/or certain lineages
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http://www.pasteur.fr/recherche/unites/Pmi
• Stewart Cole
• Stephen Gordon
• Priscille Brodin
• Daria Bottai
• Alexander Pym
• Thierry Garnier
• Tim Stinear
• Veronique Vincent
• Pedro Alzari
• Sylvain Brisse
• Julian Parkhill
Acknowledgements
• Roxane Simeone
• Alexandre Pawlik
• Alessandro Cascioferro
• Wafa Frigui
• Nadine Honore
• Mickael Orgeur
• Brigitte Saint-joanis
• Philip Supply
• Cristina Gutierrez
• Claude Leclerc
• Laleh Majlessi
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