Douglas Young

A new horizon for preventive vaccines against tuberculosis Madrid 7th May 2014

Mycobacterium tuberculosis

Evolution of Functional Diversity

844 badgers caught and sampled disease detection by serology 262 captured more than once were test negative on initial capture 22 incident cases

Chambers et al. 2011. Proc Biol Sci B. 278:1913-20 Carter et al. 2012. PLoS One 7:e49833

74% reduction in seropositive disease

79% reductionin IFN g conversion

Field trial of BCG in badgers Gloucestershire 2005-2009

group no of badgers

incident cases

% of total cases

CI F probability

control 82 14 17.1 (10.8-25.9)

vaccinated 179 8 4.5 (2.4-8.2) 0.001

unvaccinated cubs from vaccinated setts had a reduced ESAT6/CFP10 IFNg response

vaccination interrupts onward transmission

Berg et al. 2009. PLoS One 4:e5068 Firdessa et al. 2012. PLoS One 7:e52851

high prevalence > 50%

post-mortem: 67 cultures from 31 animals 67 M. bovis isolates 0 M. tuberculosis isolates

Bovine TB in Ethiopia

30000 carcasses screened in abattoirs1500 lesioned animals, 170 ZN+ cultures

low prevalence 0.5 – 5%

58 M. bovis isolates 8 M. tuberculosis isolates (12%)

A. bovine TB in rural cattle

B. bovine TB in urban intensive farms

M. tuberculosis can cause disease inindividual animals, but it doesn’t establish

an efficient transmission cycle

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?

THE CONCEPT

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

biology involved inmaking a lesion

biology involved ineffective transmission

the ideal vaccine candidate

THE MODEL

Lineage 4

Lineage 3

Lineage 2

Lineage 6 animal strains

Lineage 5

Lineage 1

Lineage 7Global phylogeny of M. tuberculosis

Comas et al. 2013. Nat Genet 45:1176

Rose et al. 2013. Genome Biol Evol 5:1849-62

Do toxin-antitoxin modules regulate “persistence”?

transcription higher in Lineage 1

transcription higher in Lineage 2

in vitro transcription profiling reveals strain variation in transcript abundance

but there’s very little evidence of genomic diversity of TA modules

0 10 20 30 40 50 60 70 80

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. phleiM. hassiacum

Mycobacterium sp. MCSM. gilvum

M. smegmatis JS623

M. chubuense

Number of TA modules

blue: chromosomered: plasmid

M avium

M. paratuberculosis

M. yongonense

M. kansasii

M. gastri

M. ulcerans

M. marinum

M. canettii 70010

M. tuberculosis

M. canettii 60008

M. xenopi

Mycobacterium sp. JDM601

M. phlei

M. hassiacum

M. smegmatis JS623

M. chubuense

M. gilvum

Mycobacterium sp. MCS

M. smegmatis MC2 155

M. abscessus

100

99

100

100

100

100

100

96

100

57

62

100

88

90

79

76

65

0.02

rpoC sequence, GTR+G+I, Maximum Likelihood phylogeny, 100 bootstrap

high TA mycobacteria (>10 modules) in redTAs and phylogeny

plasmids

lactate dehydrogenaselon protease

ddnnitroreductase

ddnnitroreductase

lactate dehydrogenase

ddnnitroreductase

lactate dehydrogenase

deletion of lon protease

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

MKAN_plasmid 29475 29470 29465 29460 29455 29450 29445 29440 29435 29430 29425 29420

MKAN_chromosome 00155 00160 00195 00200 00205 00210 00215 00220 00225

Rv1783 Rv1784 Rv1792 Rv1793 Rv1794 Rv1795 Rv1796 Rv1797 Rv1798

Rv1785 Rv1786 Rv1787 Rv1789 Rv1790 Rv1791Rv1788

eccB5 eccC5 esxM esxN eccD5 mycP5 eccE5 eccA5

cyp143 PPE25 PE18 PPE26 PPE27 PE19

PE PPE

56% 53% 91% 95% 45% 50% 55% 34% 72%

57% 52% pseudo 94% 45% 48% 57% 31% 72%

Mtb

ESX locus on pMK12478

99% identical sequence in M. yongonense plasmid pMyong1100% identical sequence in M. parascrofulaceum (plasmid?)

yrbE1A mce1A mce1B mce1C mce1D lprK mce1F Rv0175 Rv0176 Rv0177

80%

yrbE1BfadD5 Rv0178mce1R

5787 5785 5784 5783 5782 5781 5780 5779 5778 57775786 5776

60%78% 66% 63% 61% 64% 71% 52% 50% 50% 49%

5788

5775transposase transposaseM. chubuense plasmid pMYCCH01

M. tuberculosis Mce1

MCE locus on pMYCCH01

M. kansasii

M. gastri

M. ulcerans

M. marinum

M. canettii 70010

M. tuberculosis

M. canettii 60008

M. xenopi

no more horizontalgene transfer!

niche isolation?

cobF deletion

cobF

deletion in M. tuberculosis

M. canettii

M. tuberculosis

Deletion of cobF (vitamin B12) in M. tuberculosis

other methyltransferases may(partially?) compensate

Gopinath et al. 2013. Future Microbiol 8:1405

pyruvate kinase SNPalanine dehydrogenase frameshiftPhoR SNPcobL (+MK) deletion (RD9)

The Great M. tuberculosis Schism

more relaxed approachto host restriction?

increasing species adaptation?

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?

homocysteine

methionine propionyl CoA

succinate ribonucleotide

deoxyribonucleotide

MetE MetH

met

hylc

itrat

e(P

rpCD

)m

ethylmalonate

(MutAB)

NrdEF NrdZ

AMINO ACIDBIOSYTHESIS DNA REPLICATION

ENERGY

B12-independentB12-dependent

The optional metabolome of vitamin B12

Lineage 5

Lineage 6

Lineage 4

Lineage 2

Lineage 3

Lineage 7

Lineage 1

22 independentSNPs and frameshiftspredicted to impairfunction of MetH

reduced reliance onB12-dependent

pathways?

post-Neolithic?

human lung

industrial remediation

mycobacteriafreely exchanging

flexible functionality

immunologicalvomiting

nicheadaptation

transmissioncycle

no turning back(no horizontal transfer)

nicheisolation