The Genetic Structure of Mycobacterium …Genes encoding the ESX-1 type VII secretion system:...

The Genetic Structure of Mycobacterium tuberculosis

Roland Brosch

1The screen versions of these slides have full details of copyright and acknowledgements

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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

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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.


Roland Brosch


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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


Roland Brosch


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M. tuberculosis H37Rv: zoom-in

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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


Roland Brosch


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


Roland Brosch


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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


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• 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


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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


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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


Roland Brosch


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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


Roland Brosch


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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.


Roland Brosch


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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


Roland Brosch


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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



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


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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


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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)


Roland Brosch


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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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The Genetic Structure of Mycobacterium …Genes encoding the ESX-1 type VII secretion system:...

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