1. Mycobacterium tuberculosis Evolution of Functional Diversity
Douglas Young
A new horizon for preventive vaccines against tuberculosis
Madrid 7th May 2014

2. Field trial of BCG in badgers
Gloucestershire
844 badgers caught and sampled
disease detection by serology
262 captured more than once
were test negative on initial capture
22 incident cases
74% reduction
in seropositive disease
group
no of badgers
incident cases
% of total cases CI
F probability
control 82 14
17.1
( )
vaccinated
179 8
4.5
( )
0.001
unvaccinated cubs from vaccinated setts
had a reduced ESAT6/CFP10 IFNg response
79% reduction
in IFNg conversion
vaccination interrupts onward transmission
Chambers et al Proc Biol Sci B. 278:
Carter et al PLoS One 7:e49833

3. Bovine TB in Ethiopia M. tuberculosis can cause disease in
A. bovine TB in rural cattle
30000 carcasses screened in abattoirs
1500 lesioned animals, 170 ZN+ cultures
low prevalence – 5%
58 M. bovis isolates
8 M. tuberculosis isolates (12%)
B. bovine TB in urban intensive farms
high prevalence > 50%
post-mortem: 67 cultures from 31 animals
67 M. bovis isolates
0 M. tuberculosis isolates
M. tuberculosis can cause disease in
individual animals, but it doesn’t establish
an efficient transmission cycle
Berg et al PLoS One 4:e5068
Firdessa et al PLoS One 7:e52851

4. THE CONCEPT THE MODEL THE STRATEGY
I want to have a vaccine that interrupts transmission:
can I target some layer of species-specific biology
that is required for an effective transmission cycle?
biology involved in
making a lesion
effective transmission
the ideal vaccine candidate
THE MODEL
I don’t have an experimental model for transmission,
so I’m going to try and infer biology by looking at evolution of human isolates
THE STRATEGY

5. Global phylogeny of M. tuberculosis
Lineage 4
Lineage 3
Lineage 2
Lineage 6
animal strains
Lineage 5
Lineage 1
Lineage 7
Comas et al Nat Genet 45:1176

6. but there’s very little evidence of genomic diversity of TA modules
Do toxin-antitoxin modules regulate “persistence”?
transcription higher in Lineage 1
transcription higher in Lineage 2
Rose et al Genome Biol Evol 5:
in vitro transcription profiling reveals strain variation in transcript abundance
but there’s very little evidence of genomic diversity of TA modules

7. Number of TA modules blue: chromosome red: plasmid M. tuberculosis
M. canettii 60008
M. canettii 70010
Mycobacterium sp. JDM601
M. gastri
M. kansasii
M. xenopi
M. yongonense
M. paratuberculosis
M. smegmatis mc2 155
M. avium
M. marinum
M. abscessus
M. ulcerans
M. phlei
M. hassiacum
blue: chromosome
red: plasmid
Mycobacterium sp. MCS
M. gilvum
M. smegmatis JS623
M. chubuense

8. TAs and phylogeny deletion of lon protease plasmids
high TA mycobacteria (>10 modules) in red
100
M avium 88
M. paratuberculosis
lactate
dehydrogenase
lon protease
ddn
nitroreductase
deletion of lon protease 65
M. yongonense
rpoC sequence, GTR+G+I, Maximum Likelihood phylogeny, 100 bootstrap
M. kansasii 76
100
M. gastri
M. ulcerans 79
100
M. marinum
M. canettii 70010 90
100
M. tuberculosis
100 99
M. canettii 60008
M. xenopi 62
Mycobacterium sp. JDM601 96
M. phlei
M. hassiacum
plasmids 57
M. smegmatis JS623
100
M. chubuense
100
M. gilvum
Mycobacterium sp. MCS
100
M. smegmatis MC2 155
M. abscessus
0.02

9. What else is carried on mycobacterial plasmids?
toxin-antitoxin modules
metal ion detox and efflux
cytochrome P450s
adenylate cyclases
diguanylate cyclases
Type VII secretion loci
mce loci
. . .
organism
adenylate cyclase
domains
M. tuberculosis 16
M. marinum 31
M. ulcerans 15
M. smegmatis mc2 155 7
M. smegmatis JS623 48

10. ESX locus on pMK12478
MKAN_
chromosome
00155
00160
00195
00200
00205
00210
00215
00220
00225
56%
53%
91%
95%
45%
50%
55%
34%
72%
MKAN_
plasmid
29475
29470
29465
29460
29455
29450
29445
29440
29435
29430
29425
29420 PE
PPE
57%
52%
pseudo
94%
45%
48%
57%
31%
72%
Mtb
Rv1783
Rv1784
Rv1792
Rv1793
Rv1794
Rv1795
Rv1796
Rv1797
Rv1798
eccB5
eccC5
esxM
esxN
eccD5
mycP5
eccE5
eccA5
Rv1785
Rv1786
Rv1787
Rv1788
Rv1789
Rv1790
Rv1791
cyp143
PPE25
PE18
PPE26
PPE27
PE19
99% identical sequence in M. yongonense plasmid pMyong1
100% identical sequence in M. parascrofulaceum (plasmid?)

11. MCE locus on pMYCCH01 M. chubuense plasmid pMYCCH01
transposase
5788
M. chubuense plasmid pMYCCH01
transposase
5775
5787
5786
5785
5784
5783
5782
5781
5780
5779
5778
5777
5776
80%
78%
60%
66%
63%
61%
64%
71%
52%
50%
50%
49%
mce1R
fadD5
yrbE1A
yrbE1B
mce1A
mce1B
mce1C
mce1D
lprK
mce1F
Rv0175
Rv0176
Rv0177
Rv0178
M. tuberculosis Mce1

12. no more horizontal gene transfer!
niche isolation?
M. kansasii
M. gastri
M. ulcerans
M. marinum
M. canettii 70010
M. tuberculosis
M. canettii 60008
M. xenopi
cobF deletion

13. Deletion of cobF (vitamin B12) in M. tuberculosis
M. canettii
deletion in M. tuberculosis
M. tuberculosis
other methyltransferases may
(partially?) compensate
Gopinath et al Future Microbiol 8:1405

14. The Great M. tuberculosis Schism
pyruvate kinase SNP
alanine dehydrogenase frameshift
PhoR SNP
cobL (+MK) deletion (RD9)
The Great M. tuberculosis Schism
more relaxed approach
to host restriction?
increasing
species adaptation?

15. M. tuberculosis may have evolved
to rely on vitamin B12 provided by the host?
niche adaptation
bioavailability of B12 in primates versus ruminants?
effect of diet – vegetarian versus meat-eating?
gut microbiome?

16. The optional metabolome of vitamin B12
AMINO ACID
BIOSYTHESIS
DNA REPLICATION
methionine
propionyl CoA
deoxyribonucleotide
methylcitrate
(PrpCD)
methylmalonate
(MutAB)
MetE
MetH
NrdEF
NrdZ
homocysteine
succinate
ribonucleotide
B12-independent
B12-dependent
ENERGY

17. 22 independent SNPs and frameshifts predicted to impair
Lineage 5
Lineage 6
22 independent
SNPs and frameshifts
predicted to impair
function of MetH
Lineage 4
Lineage 2
reduced reliance on
B12-dependent
pathways?
Lineage 3
Lineage 7
Lineage 1
post-Neolithic?

18. niche adaptation transmission cycle niche isolation
immunological
vomiting
niche
adaptation
human lung
transmission
cycle
no turning back
(no horizontal transfer)
niche
isolation
mycobacteria
freely exchanging
flexible functionality
industrial remediation