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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

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M. tuberculosisM. tuberculosis derived derived from from M. bovisM. bovis

M. bovis

M. tuberculosis

Or was it?

Proposed originProposed origin

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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

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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

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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

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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’

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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.

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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

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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

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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

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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

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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

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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

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Evolution of the Evolution of the M. tbM. tb complex complex

M. bovis

M. tuberculosis

X

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Progenitor bacillus

M. bovis M. tuberculosis

Evolution of the Evolution of the M. tbM. tb complexcomplex

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Has Has M. tbM. tb evolved since? evolved since?

Different approaches to population genetics

All based on genomics

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Mycobacterium canettii Mycobacterium canettii isis smoothsmooth

M. tuberculosis

M. canettii

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Split decomposition analysis, SNP dataSplit decomposition analysis, SNP data

MTBC(worldwide)

Smooth tubercle bacilli

(Djibouti, East Africa)

M. canettii

M. prototuberculosis

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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.

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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.

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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

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SNP typing - 3 SNP typing - 3

Studied 159 sSNPs in 219 isolates

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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

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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

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Beijing phylogenyBeijing phylogeny

Marmiesse et al. (2004) Microbiology 150: 483 - 496

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A new lipid - PGL - in BeijingA new lipid - PGL - in Beijing

Reed et al. (2004) Nature 431: 84-87

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Effect of PGL on virulenceEffect of PGL on virulence

Reed et al. (2004) Nature 431: 84-87

Immunocompetent mice, aerosol infection

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Immunologic effects of PGLImmunologic effects of PGL

Reed et al. (2004) Nature 431: 84-87

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Further immunologic effectsFurther immunologic effects

Reed et al. (2004) Nature 431: 84-87

Single sugar accounts for difference

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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

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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

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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