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

Field trial of BCG in badgers Gloucestershire 2005-2009 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 (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 79% reduction in IFNg conversion vaccination interrupts onward transmission Chambers et al. 2011. Proc Biol Sci B. 278:1913-20 Carter et al. 2012. PLoS One 7:e49833

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 0.5 – 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. 2009. PLoS One 4:e5068 Firdessa et al. 2012. PLoS One 7:e52851

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

Global phylogeny of M. tuberculosis Lineage 4 Lineage 3 Lineage 2 Lineage 6 animal strains Lineage 5 Lineage 1 Lineage 7 Comas et al. 2013. Nat Genet 45:1176

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. 2013. Genome Biol Evol 5:1849-62 in vitro transcription profiling reveals strain variation in transcript abundance but there’s very little evidence of genomic diversity of TA modules

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

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

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

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?)

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

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

Deletion of cobF (vitamin B12) in M. tuberculosis M. canettii deletion in M. tuberculosis M. tuberculosis other methyltransferases may (partially?) compensate Gopinath et al. 2013. Future Microbiol 8:1405

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?

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?

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

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?

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