On the evolution of
pathogenic mycobacteria
(and their antigens)
Stewart Cole
1
Phylogenetic tree - slow growers
M. fortuitum
M. peregrinum
M. triviale
M. simiae
M. genavense
MCRO18
MCRO19
M. interjectum
M. intermedium
Springer et al., J. Clin. Microbiol.,
(1996) 34:296-303
2
M. terrae
M.
M.
hiberniae
MCRO6
nonchromogenicu
M. cookii
M. celatum
m
M. xenopi
M. shimoidei
M. tuberculosis
M. asiaticum
M. africanum
M. gordonae
M.canettii
M. marinum
M. ulcerans
M. microti
M. tuberculosis
M. bovis
M. leprae
M. Complex
szulgai
M. bovis BCG
M. malmoense
M. haemophilum
M. gasti / kansasli
M. scrofulaceum
M.
M.
intracellulare»
« paraffinicum
M.
M. avium
paratuberculosis
Tuberculosis
Leprosy
Mycobacterial cell envelope
3
Genome of M. tuberculosis
4,000 genes
40%
orphans
Cole et al. (1998)
Nature 393: 537-544
4
Maps of
other spp.
nearly identical
Duplicated proteins in M. tb.
Tekaia et al. (1999) Tuber Lung Dis 79, 329-342.
200
PE & PPE proteins
Lipid metabolism
ABC-transporters, etc
Transcriptional regulators
100
ESAT-6
0
Number of genes
5
Unanswered questions
• PE, PPE & ESX gene families occupy 10% of
genome
• What do they do?
• Involved in pathogenesis &/or persistence?
• Immune evasion, antigenic variation?
6
Domain structure of PE & PPE proteins
94-110 aa
PE
170-588 aa
PE
Unique sequence
40-1680 aa
PE
~ 180 aa
PPE
PGRS
MPTR
GxxSVPxxW
0-400 aa
PPE
7
(GGAGGAGGN)n
200-3500 aa
200-400 aa
PPE
7-314 aa
Unique
(NXGXGNXG)n
Some PE-PGRS surface-located
& variable
Immunogold EM
Banu et al. 2002
Mol. Microbiol.
44: 9-19
8
Western blot
cell envelope
proteins from
clinical isolates
Variability in PE genes
M. bovis 2122/97
193 aa
1370
126 aa
162 aa
1776
1887 aa
(a) Rv1917c
M. tuberculosis H37Rv
1436 aa
427
M. bovis 2122/97
482
549
23 aa
1250 4 aa
50 aa
1051 aa
(b) Rv1753c
M. tuberculosis H37Rv
52 aa
9
850
1053 aa
PE_PGRS family
Same thing seen
with PPE_MPTR
PGRS gene conversion?
Rv1450
+9
Rv1451Rv1452
∆9
S21
Mb1485
Mt1497
∆9
∆69
225
PE
295 364
Rv1450
764
1284
PGRS
295 345 450
475
S21
501
+3
S33∆4 S11 S10∆3
S11 S10∆3
10
Orthologs
Homogeno
-tisation
686
Mb1487
+48
Mt1499
Rv1452
Orthologs
Impact on innate immunity?
In-frame deletion-variants have altered effects
11
PE-PGRS inhibit proteasomes
A
B
C
Brennan et al. (2002)
TIMS 10, 246
12
What is the role of the PE-PGRS?
• Purely structural
• Innate immunity via TLR2
• Variable surface antigen
• Immunological smoke-screen
• Proteasome inhibitor, cf EBNA-1
• Blocks ag presentation by MHC class I
13
Immunogenicity islands
PE35 PPE68
100
330
Envelope
CHP, chaperones,
transporters
T-cell antigens
PE66 PPE83
100
14
330
EsxB EsxA
95
98
Major, secreted
T-cell antigens
EsxX EsxY
95
N=5
98
CHP, 11TM,
S-proteases
N=6
RD1 in M. bovis BCG: ESX-1
RD1mic
Rv3860
Rv3866 Rv3868 Rv3869
RD1BCG
Rv3864/65
Rv3861Rv3863
Rv3876
PE/PPE
esx
Rv3867
Rv3870
Rv3871
Rv3874
Rv3877Rv3878
4336 4337 4338 4339 4340 4341 4342 4343 4344 4345 4346 4347 4348 4349 4350 4351 4352 4353 4354 4355 4356 4357 4358 4359 4360 4361 4362 4363 4364 4365 4366 4367 4368 4369 4370 4371 4372
Rv3884
Rv3879
Rv3862
Rv3880
Rv3882
Rv3881
4,340,417
ESAT-6 locus
of Tekaia et al.
15
4,350,263
Rv3872
Rv3873
Rv3874
Rv3875
Rv3876
Rv3877
Rv3878
Rv3879
PE
PPE
CFP10
ESAT-6
4,354,536
Rv3885
Rv3883
4,359,716
Secreted T cell antigen
Secreted T cell antigen
ATPase
Integral membrane protein
Unknown
Unknown-Proline rich N-terminal
Rv3886
Rv3887
ESX-1 increases virulence in SCID mice
M. bovis
BCG Pasteur
BCG::RD1-2F9
+RD1
BCG
M. microti
OV254
OV254::RD1-2F9
16
Model for ESX-1 function
Secretes major T-cell antigens
Required for uptake & cell-cell spread
Pym et al. (2002) Mol Microbiol 46: 709;
Pym et al. (2003) Nat Med 14: 533;
Brodin et al. (2004) J Inf Dis 190: 115;
Brodin et al. (2005) J Biol Chem. 280: 33953
Renshaw et al. (2005) EMBO J. 24: 2491-8.
17
M. leprae - genome decay
Slide 12
18
Comparative genomics: M. tb - M. leprae
Mycobacterium tuberculosis
Alpha, keto
& methoxymycolates
trpT secA
nusG rplK rplA
mmaA4
lipG
mmaA1
Rv0647
mmaA3 mmaA2
Mycobacterium leprae
No methoxymycolates
nusG rplK
trpT secA
rplA
pseudo
MLCB41.12
mmaA4
pseudo
19
mmaA1
pseudo
lipG
MLCB41.06
Gross features of M. leprae genome
• 1,605 genes
M. leprae has
• 1,433 genes common undergone
with M. tub.
• >1 ,116 pseudogenes
reductive evolution
• ~1,500 genes "deleted
"
Rep-DNA
involved
• ~160 M. lep "specific" genes
• Mosaic, ~65 segments = M. tub
20
Main IS & repeats in M. leprae
• 2% of genome made of repeats
• 26 different IS, all defective
• RLEP (37 copies, > 1 truncated, ~850 bp)
• REPLEP (15 copies, 2 truncated, 881 bp)
• LEPREP (8 copies, 3 truncated, 2383 bp)
• LEPRPT (5 copies, 2 truncated, 1252 bp)
• None has ORFs, some have IR
21
What happened to synteny?
Recombination between rep-DNA
Rv1056
ML 0266
22
Rv0409, Rv0408
M. tuberculosis
(ackA, pta)
REPLEP
ML0267, ML0268
M. leprae
Deletions mediated by REPLEP?
xseAB
Rv1105,04
Rv1100
Rv1103,02,01
Rv1099, fum
Rv1106
M. tuberculosis
M. leprae then
REPLEP
REPLEP
M. leprae later
REPLEP
M. leprae today
xseAB ML1942
23
ML1946, fum
ML1945
Depleted PE/PPE repertoire in M. leprae
Number within Category
400
1000
800
350
600
300
400
M. leprae genes
M. leprae pseudogenes
M. tuberculosis genes
200
250
0
29
200
30
150
100
50
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14
15 16 17 18
Functional Category
24
19
20 21 22 23
24 25 26 27 28
Esx repertoire
reduced.
Esx1, 3
2(PE,PPE,Esx2)
Mycobacterial phylogeny
M. fortuitum
M. peregrinum
M. triviale
M. simiae
M. genavense
M. ulcerans
MCRO18
MCRO19
M. interjectum
M. intermedium
M. terrae
M. hiberniae
M. nonchromogenicum
MCRO6
M. cookii
M. celatum
M. marinum
25
M. shimoidei
M. asiaticum
M. gordonae
M. marinum
M. ulcerans
M. tuberculosis
M. leprae
Complex
M. szulgai
M. malmoense
M. haemophilum
M. gastri / kansasi
M. scrofulaceum
M. « paraffinicum »
M. intracellulare
M. paratuberculosis
M. avium
M. xenopi
Buruli ulcer - an emerging disease
• No therapy
• No vaccine
• Surgery & grafts
• Painless, no IR
• Not transmissible
between humans
26
Mycolactone
Rapamycin
Mycolactone - an assembly-line product
27
Transmission of M. ulcerans
Naucoris
cimicoides
Marsollier, L., et al.
(2002) Aquatic insects
as a vector for
Mycobacterium ulcerans.
Appl Environ Microbiol
68, 4623-8.
28
Larvae?
Molluscs?
Fish?
Ducks?
PCR +
PCR +
culture
PCR +
The M. ulcerans genome
ISEs
M. ulcerans Agy99
5,631,606 bp
Genes specific to
29
M. ulcerans (30)
Genes common to all
mycobacteria (1000)
First mycobacterial virulence plasmid
Stinear et al. 2004
PNAS 101: 1345
30
Giant PKS produce mycolactone
Locus arose by duplication
16 tandem repeats
31
Bipartite whole genome comparison
>98% sequence ID
213 x IS2404, 91 x IS2606
32
Stinear et al. (2007)
Genome Res 17: 192-200.
Some genome statistics
33
M. marinum
M. ulcerans
Chromosome size
6,636,827
5,631,606
Genes
5,434
4,160
Pseudogenes
52
771
% GC
65.73
65.71
Gen. Time (h)
4
36
Depletion of PE, PPE, ESX repertoire
M. marinum M. ulcerans
PE
170
70
PPE
105
46
Favors extracellular growth?
ESX
24
13
Ext-ESX
5
3
Pseudos
0
109
34
Where did M. ulcerans come from?
• Derived from MM after acquiring
plasmid with genes for mycolactone
• But pMUM001 may be mosaic…
http://genstyle.imed.jussieu.fr/
…. and have introduced IS elements
35
Evolutionary driving forces?
• Environmental ancestor, large genome:
Lots of PE, PPE, ESX?
• Change in environment:
Stable niche, genome degenerates, rep-DNA involved
• In host, lose PGRS, ESX etc.
Selection for less immunogenic, less virulent variants?
36
With the participation of...
Institut Pasteur
S. Banu
R. Brosch
K. Eiglmeier
T. Garnier
N. Honoré
L. Marsollier
G. Meurice
M. Monot
A.S. Pym
G. Reysset
T. Stinear
37
Collaborators
B.G. Barrell
S.V. Gordon
R.G. Hewinson
J. Parkhill
L. Ramakrishnan
P.L.C. Small
NIH
NIAID
ILEP
AFRF