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Evolutionary genomics Evolutionary genomics of mycobacterial of mycobacterial pathogens - 2pathogens - 2(On the origin of(On the origin oftuberculosis)tuberculosis)
Stewart ColeStewart Cole
2
M. tuberculosisM. tuberculosis derived derived from from M. bovisM. bovis
M. bovis
M. tuberculosis
Or was it?
Proposed originProposed origin
3
Recent evolution of TB Recent evolution of TB bacillibacilli
Proc. Natl. Acad. Sci. USAVol. 94, pp. 9869-74, September 1997Genetics
Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global disseminationS. Sreevatsan, X. Pan, K.E. Stockbauer, N.D. Connell, B.N. Kreiswirth, T.S. Whittam AND J.M. MusserSection of Molecular Pathobiology, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Communicated by B.R. Bloom, Albert Einstein College of Medicine, Bronx, NY, July 4, 1997 (received for review May 6, 1997)
ABSTRACT One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of approximately 6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.
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Genomics of tubercle bacilliGenomics of tubercle bacilli
M. tuberculosis complex
AF2122/97Shotgunfinished
ShotgunH37RvCDC1551K- strain
M. tuberculosis M. africanumM. canettii M. microti M. bovis M. bovis BCG
BCG-Pasteur
Finished
In progress
4.41 Mb 4.32 Mb
4.31Mb
Shotgun
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Maps of other spp.
nearly identical
4,000 genes 40%
orphans
Genome of Genome of M. tuberculosisM. tuberculosis
Cole et al. (1998) Nature 393: 537-544
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Sources of genetic Sources of genetic diversitydiversity
• Point mutations or SNP
• InDels
• Insertions: IS, gene dup, HT,
replication errors
• Deletions: RecA, IS-mediated,
replication errors
• Translocations
PZA-R
IS6110, BCG
Common, RD
None to date
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Evolutionary Genomics of TB BacilliEvolutionary Genomics of TB Bacilli
8
Comparative genomic statisticsComparative genomic statistics
Differences H37Rv: M.bovis
CDC1551: M.bovis
H37Rv:CDC1551
SNP 2346 2380 1135Deletions 203 213 281Insertions 173 104 63
Substit. 160 247 166
InDels drive plasticity
TbD1: Major region of difference
between Mt & Mb
Garnier et al. (2003) PNAS 100:7877
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TbD1 truncates MmpL6TbD1 truncates MmpL6
∆ M. tuberculosis
Might affectlipid/glycolipid
export
10
RD9 - an ancient RD9 - an ancient deletiondeletion
M. africanum
M. microti
BCG
M. bovis
AAATTACTGTGGCCCACGCCGGGCCGG
M. tuberculosis
Rv2073c Rv2074 Rv2075c
..TTGGTGGCACGCCGGGCCGG
AAATTACTGTGGCCCTGCGCAA....
cobL
Cannot be due to insertion
M. tuberculosis
H37Ra
M. tuberculosis
H37Rv
Rv1758 ’IS6110 RvD2-ORF1
RvD2-ORF2 RvD2-ORF3
plcD ’ plcD ’
Rv1758 ’
IR
IR
IR
IR
IS6110 IS6110
IS6110
Rv1758 ’
IS6110
IR
IS6110plcD ’
Rv1758 ’
IR
Rv1758 M. bovis
RvD2-ORF3RvD2-ORF2
plcD RvD2-ORF1
GAG AGC
AGCGAG
Lessinformative
RvD2 - a recent deletionRvD2 - a recent deletion
12
RD regions in M. tb complexRD regions in M. tb complex
RD5 (mpt40)
Rv 2345
plcC plcB plcA PPE PPE
IS61102625 2626 2627 2628 2629 2630 2631 2632 2633 2634 2635 26362624 kb
Rv 2346/47/48
ephA Rv3618 lpqG
Rv3616 Rv3619/20 PPE PE
RD8
4061 4062 406340604059405840574056 kb
RD10
Rv0221 echA1
Rv0223
0265 0266 0267 02680264
Rv0224
kb
RD9
Rv2075/76
Rv2074
Rv2073cobLcobM
2330 23332329 23322331 kb
RD12
3483 3484 3485 3486 3487 3488 3489moeB
3490 kbcys3A
sseCmoaE
Rv3120/21/22/23 Rv3124RD13
1401 1402 1403 1404 1405 1406deaD
1407 kbRv1254
Rv1255
Rv1256 Rv1257 Rv1258RD11 (phiRv2)
Rv2645/46/47 IS6110
Rv2650/51 Rv2652/53/54Rv2655/56/57/58/59/60/61
glyT cysTvalU
valTRv2644
2970 2971 2972 2973 2974 2675 2976 2977 2978 2979 2980 2981 kb2969
RD3 (phiRv1)
REP ’
Rv1573/74/75
Rv1576/77/78Rv1579/80/81Rv1582/83/84/85 / 86 REP
bioB17801779 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 kb
bioD 1778
Rv1571
oriC
Mycobacterium bovis BCG Pasteur 1173 P2
PPEPPEPPE
Rv3424alr IS1532
RD6
3841 38443842 3843 3845 3846 3847 3848kb
Rv3430
RD1
PE/PPE4359 4360 4361 kb4351 43544352 4353 4355 4356 4357 43584349 4350
Rv3871 Rv3874esat6Rv3876 Rv3878
Rv3879 Rv3880/81
Rv3877
Rv1771200019991998 2001 2002 2003 2004 2005 2006 2007 2008 2009 kb
Rv1766/67
IS9’Rv1765
Rv1770Rv1769PE_PGRS
Rv1772Rv1774
Rv1773
RD14
RD4
gmdAepiA Rv1513
Rv1514/15 Rv1516
Rv1517
Rv1508
Rv1509Rv151017001699169816971696 1701 1702 1703 1704 1705 1706 1707 1708 1709 kb
Rv1505/06/07
kb
RD7 RD2
Rv1965Rv1964 mce3 Rv1967Rv1968Rv1969lprM Rv1971/72/73/74/75
Rv1976
Rv1977 Rv1978
Rv1979mpt64 nrdFRv1982
PE-PGRS
Rv1984 Rv1985
Rv1986Rv1987/882208 22092207 22202219221822172216221522142213221222112210 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232
Rv1963 Rv1989
RD9 ishere!
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RD distribution in M. tbcRD distribution in M. tbc
M. tub. M. afri. M. mic. M. bov. BCG
RD 9
RD3 ( Rv1)
RD 9RD 7RD 8RD 10
RD3 ( Rv1)RD 5’
RD 9RD 7RD 8RD10
RD 4RD 5RD12RD13
RD 9RD 7RD 8RD10
RD 4RD 5RD12RD13
RD 1RD 2
RD3 ( Rv1)
RD11 ( Rv2)
RD11 ( Rv2)
M. can.
TbD1RD 12’
14
oxyR 285 GA
Common ancestor of the M. tuberculosis complex
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tuberculosiskatG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal-isol.oryx-isol.
goat-isol.
“classical”RD 1
BCG Tokyo
gyrA95AGCACC
pncA 57CACGAC
RD 4
RD 2
BCG Pasteur
RD 14
TbD 1
Numerous sequence polymorphisms
“modern”
“ancestral”
mmpL6 551AACAAG
Evolutionary scenarioEvolutionary scenario
Brosch et al. 2002 Proc Natl Acad Sci U S A.
99:3684-9.
15
oxyR 285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal
oryx
goat
“classical”RD 1
BCG Tokyo
gyrA 95AGCACC
pncA57CACGAC
RD 4
RD 2
BCG PasteurRD 14
TbD 1
“modern”
“ancestral”
RD9 +
mmpL6 551AACAAG
Rapid ID of TB bacilliRapid ID of TB bacilli
16
oxyR n285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9
M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal
oryx
goat
“classical”RD 1
BCG Tokyo
gyrA 95AGCACC
pncAc57CACGAC
RD 4
RD 2
BCG PasteurRD 14
TbD 1
“modern”
“ancestral”
RD9 +
TbD1 -mmpL6 551AACAAG
eg. Beijing cluster
eg. Haarlem cluster
eg. H37Rv
Rapid ID of TB bacilliRapid ID of TB bacilli
17
oxyR n285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal-isolates
oryx-isolates
goat-isolates
“classical”RD 1
BCG Tokyo
gyrA 95AGCACC
pncAc57CACGAC
RD 4
RD 2BCG Pasteur
RD 14
TbD 1
“modern”
“ancestral”
RD9 -
mmpL6 551AACAAG
Rapid ID of TB bacilliRapid ID of TB bacilli
18
oxyR n285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
“classical”RD 1
BCG Tokyo
gyrA 95AGCACC
pncA 57CACGAC
RD 4
RD 2BCG Pasteur
RD 14
TbD 1
“modern”
“ancestral”
RD9 -
mmpL6 551AACAAG
mmpL6 551 AAG
seal-isolates
oryx-isolates
goat-isolates
Rapid ID of TB bacilliRapid ID of TB bacilli
19
oxyR n285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal
oryx
goat
“classical”RD 1
BCG Tokyo
gyrA 95AGCACC
pncA 57CACGAC
RD 4
RD 2BCG PasteurRD 14
TbD 1
“modern”
“ancestral”
RD9 -
mmpL6 551AACAAG
RD4 -
Rapid ID of TB bacilliRapid ID of TB bacilli
20
oxyR n285 GA
M. africanum
RD 7RD 8
RD 10
RD 12
RD 13
M. canettii
RD 9M. tub.katG 463 CTGCGG
M. microti
M. bovis
RDcan
RDmic
RDseal
seal
oryx
goat
“classical”RD 1
BCG
gyrA 95AGCACC
pncA 57CACGAC
RD 4
RD 2
RD 14
TbD 1
“modern”
“ancestral”
RD9 -
mmpL6 551AACAAG
RD1 -
Rapid ID of TB bacilliRapid ID of TB bacilli
21
Evolution of the Evolution of the M. tbM. tb complex complex
M. bovis
M. tuberculosis
X
22
Progenitor bacillus
M. bovis M. tuberculosis
Evolution of the Evolution of the M. tbM. tb complexcomplex
23
Has Has M. tbM. tb evolved since? evolved since?
Different approaches to population genetics
All based on genomics
24
Mycobacterium canettii Mycobacterium canettii isis smoothsmooth
M. tuberculosis
M. canettii
25
Split decomposition analysis, SNP dataSplit decomposition analysis, SNP data
MTBC(worldwide)
Smooth tubercle bacilli
(Djibouti, East Africa)
M. canettii
M. prototuberculosis
26
LSP (RD) typing LSP (RD) typing
Gagneux et al. (2006) Variable host-pathogen compatibility in M. tuberculosis. Proc Natl Acad Sci
U S A; 103: 2869-2873.
27
SNP typing - 1 SNP typing - 1
Examined 37 sSNPs in 225 isolates
Baker et al. (2004) Silent nucleotide polymorphisms and
a phylogeny for Mycobacterium tuberculosis. Emerg Infect Dis 2004; 10: 1568-77.
28
SNP typing - 2 SNP typing - 2
Gutacker et al. (2006) Single-nucleotide polymorphism-based
population genetic analysis of Mycobacterium tuberculosis strains
from 4 geographic sites. J Infect Dis; 193: 121-128.
36 sSNPs in 5069 isolates
29
SNP typing - 3 SNP typing - 3
Studied 159 sSNPs in 219 isolates
30
Global distribution Global distribution
Red Euro-AmericanGreen W-African 1Brown W-African 2Yellow Indo-OceanicYellow Indo-Oceanic
Purple EA-IndianBlue East Asian
Blue is mostworrying
31
The Beijing familyThe Beijing family
Appears to be more virulent, more transmissible &
associated with MDR
TRENDS in Microbiology Vol.10 No.1 January 2002
45-52
32
Beijing phylogenyBeijing phylogeny
Marmiesse et al. (2004) Microbiology 150: 483 - 496
33
A new lipid - PGL - in BeijingA new lipid - PGL - in Beijing
Reed et al. (2004) Nature 431: 84-87
34
Effect of PGL on virulenceEffect of PGL on virulence
Reed et al. (2004) Nature 431: 84-87
Immunocompetent mice, aerosol infection
35
Immunologic effects of PGLImmunologic effects of PGL
Reed et al. (2004) Nature 431: 84-87
36
Further immunologic effectsFurther immunologic effects
Reed et al. (2004) Nature 431: 84-87
Single sugar accounts for difference
37
PGL impacts on phenotype PGL impacts on phenotype
Reed et al. (2004) Nature 431: 84-87
•Increases lethality greatly but not bacterial load
•Down-regulates pro-inflammatory response in
dose-dependent manner
•Represses TNF-alpha, IL-6 & IL-12 production
•May contribute to increased transmission
38
SummarySummary
M. tuberculosis complex tightly knit but differences in host range
M. tuberculosis not descended from M. bovis but possibly from M. prototuberculosis
Species became host adapted. 4-5 major M.tb groups
Hypervirulent variants emerge and replace existing clones
39
With the participation With the participation of...of...
ILEP
Institut PasteurInstitut PasteurR. BroschR. BroschS. BrisseS. Brisse
M-C. GutierrezM-C. GutierrezT. GarnierT. GarnierN. HonoréN. Honoré
M. MarmiesseM. MarmiesseV. VincentV. Vincent
WT Sanger InstituteWT Sanger InstituteB.G. BarrellB.G. BarrellJ. ParkhillJ. Parkhill
M-A. RajandreamM-A. Rajandream
Central Veterinary Lab.Central Veterinary Lab.R.G. HewinsonR.G. Hewinson
S.V. GordonS.V. Gordon
NIHNIAID