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On the evolution of pathogenic mycobacteria (and their antigens)

On the evolution of pathogenic mycobacteria (and their antigens). Stewart Cole. M. fortuitum. M. peregrinum. M. triviale. M. simiae. M. genavense. MCRO18. MCRO19. M. interjectum. M. intermedium. M. terrae. M. hiberniae. M. nonchromogenicum. MCRO6. M. cookii. M. celatum. M. xenopi.

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On the evolution of pathogenic mycobacteria (and their antigens)

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  1. On the evolution of pathogenic mycobacteria(and their antigens) Stewart Cole

  2. M. fortuitum M. peregrinum M. triviale M. simiae M. genavense MCRO18 MCRO19 M. interjectum M. intermedium M. terrae M. hiberniae M. nonchromogenicum MCRO6 M. cookii M. celatum M. xenopi M. shimoidei M. asiaticum M. gordonae M. marinum M. ulcerans M. tuberculosis Complex M. leprae M. szulgai M. malmoense M. haemophilum M. gasti / kansasli M. scrofulaceum M. « paraffinicum » M. intracellulare M. paratuberculosis M. avium Phylogenetic tree - slow growers Tuberculosis M. tuberculosis M. africanum M.canettii M. microti M. bovis M. bovis BCG Leprosy Springer et al., J. Clin. Microbiol., (1996) 34:296-303

  3. Mycobacterial cell envelope

  4. Genome of M. tuberculosis 4,000 genes 40% orphans Maps of other spp. nearly identical Cole et al. (1998) Nature 393: 537-544

  5. Duplicated proteins in M. tb. PE & PPE proteins Lipid metabolism ABC-transporters, etc Transcriptional regulators ESAT-6 Tekaia et al. (1999) Tuber Lung Dis 79, 329-342. 200 Number of partitions 100 0 Number of genes

  6. Unanswered questions • PE, PPE & ESX gene families occupy 10% of genome • What do they do? • Involved in pathogenesis &/or persistence? • Immune evasion, antigenic variation?

  7. PE PE PE PPE PPE PPE Domain structure of PE & PPE proteins 94-110 aa 170-588 aa Unique sequence 40-1680 aa 7-314 aa (GGAGGAGGN)n PGRS ~ 180 aa 200-3500 aa (NXGXGNXG)n MPTR 200-400 aa GxxSVPxxW 0-400 aa Unique

  8. Some PE-PGRS surface-located & variable Immunogold EM Western blot cell envelope proteins from clinical isolates Banu et al. 2002 Mol. Microbiol. 44: 9-19

  9. Variability in PE genes M. bovis 2122/97 1370 1776 193 aa 126 aa 162 aa 1887 aa (a) Rv1917c M. tuberculosis H37Rv 1436 aa 1250 427 549 23 aa 4 aa M. bovis 2122/97 482 50 aa 1051 aa (b) Rv1753c M. tuberculosis H37Rv 1053 aa 850 52 aa PE_PGRS family Same thing seen with PPE_MPTR

  10. PGRS gene conversion? +9 S21 Mb1487 Mb1485 Rv1452 Rv1450 Rv1451 Mt1497 Mt1499 ∆9 Rv1452 Rv1450 Orthologs ∆9 ∆69 225 295 364 764 1284 Homogeno -tisation PE PGRS 295 345 450 501 686 475 Orthologs S21 +3 +48 S33∆4 S11 S10∆3 S11 S10∆3

  11. Impact on innate immunity? In-frame deletion-variants have altered effects

  12. PE-PGRS inhibit proteasomes B A C Brennan et al. (2002) TIMS 10, 246

  13. 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

  14. Immunogenicity islands PE66 PE35 PPE68 PPE83 EsxX EsxB EsxA EsxY CHP, chaperones, transporters CHP, 11TM, S-proteases 100 100 330 330 95 95 98 98 N=5 Envelope T-cell antigens Major, secreted T-cell antigens N=6

  15. RD1mic RD1BCG 4,340,417 4,354,536 4,359,716 4,350,263 RD1 in M. bovis BCG: ESX-1 Rv3876 Rv3860 Rv3866 PE/PPE Rv3868 Rv3869 Rv3864/65 esx Rv3874 Rv3877 Rv3878 Rv3861 Rv3867 Rv3863 Rv3870 Rv3871 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 Rv3885 Rv3862 Rv3882 Rv3886 Rv3880 Rv3881 Rv3883 Rv3887 ESAT-6 locus of Tekaia et al. Rv3872 PE Rv3873 PPE Rv3874 CFP10 Secreted T cell antigen Rv3875 ESAT-6 Secreted T cell antigen Rv3876 ATPase Rv3877 Integral membrane protein Rv3878 Unknown Rv3879 Unknown-Proline rich N-terminal

  16. M. bovis BCG Pasteur BCG::RD1-2F9 M. microti OV254 +RD1 OV254::RD1-2F9 BCG ESX-1 increases virulence in SCID mice

  17. 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.

  18. M. leprae - genome decay Slide 12 Slide 12

  19. Comparative genomics: M. tb - M. leprae Mycobacterium tuberculosis Alpha, keto & methoxy- mycolates secA trpT nusG rplK rplA mmaA4 lipG mmaA1 Rv0647 mmaA3 mmaA2 Mycobacterium leprae No methoxy- mycolates nusG rplK secA trpT rplA pseudo MLCB41.12 mmaA1 lipG mmaA4 pseudo MLCB41.06 pseudo

  20. Gross features of M. leprae genome • 1,605 genes • 1,433 genes common with M. tub. • >1,116 pseudogenes • ~1,500 genes "deleted "    • ~160 M. lep "specific" genes • Mosaic, ~65 segments = M. tub M. leprae has undergone reductive evolution Rep-DNA involved

  21. 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

  22. What happened to synteny? Recombination between rep-DNA Rv1056 Rv0409, Rv0408 (ackA, pta) M. tuberculosis ML0266 REPLEP ML0267, ML0268 M. leprae

  23. Rv1105,04 Rv1100 M. tuberculosis Rv1103,02,01 Rv1099, fum xseAB Rv1106 REPLEP REPLEP M. leprae later REPLEP M. leprae today xseAB ML1942 ML1946, fum ML1945 Deletions mediated by REPLEP? M. leprae then

  24. 1000 800 600 400 400 M. leprae genes 200 350 M. leprae pseudogenes M. tuberculosis genes 0 29 30 300 250 Number within Category 200 150 100 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 50 0 Functional Category Depleted PE/PPE repertoire in M. leprae Esx repertoire reduced. Esx1, 3 2(PE,PPE,Esx2)

  25. M. fortuitum M. peregrinum M. triviale M. simiae M. genavense MCRO18 M. ulcerans MCRO19 M. interjectum M. intermedium M. terrae M. hiberniae M. nonchromogenicum MCRO6 M. cookii M. celatum M. xenopi M. shimoidei M. asiaticum M. gordonae M. marinum M. ulcerans M. tuberculosis Complex M. leprae M. szulgai M. malmoense M. haemophilum M. gastri / kansasi M. scrofulaceum M. « paraffinicum » M. marinum M. intracellulare M. paratuberculosis M. avium Mycobacterial phylogeny

  26. No therapy • No vaccine Buruli ulcer - an emerging disease • Surgery & grafts • Painless, no IR • Not transmissible between humans

  27. Mycolactone Rapamycin Mycolactone - an assembly-line product

  28. Transmission of M. ulcerans PCR + PCR + PCR + culture Naucoris cimicoides Larvae? Molluscs? Fish? Ducks? Marsollier, L., et al. (2002) Aquatic insects as a vector for Mycobacterium ulcerans. Appl Environ Microbiol 68, 4623-8.

  29. ISEs M. ulcerans Agy99 5,631,606 bp Genes specific to M. ulcerans (30) Genes common to all mycobacteria (1000) The M. ulcerans genome

  30. First mycobacterial virulence plasmid Stinear et al. 2004 PNAS 101: 1345

  31. Giant PKS produce mycolactone Locus arose by duplication 16 tandem repeats

  32. Bipartite whole genome comparison >98% sequence ID 213 x IS2404, 91 x IS2606 Stinear et al. (2007) Genome Res 17: 192-200.

  33. Some genome statistics 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

  34. Depletion of PE, PPE, ESX repertoire M. marinum M. ulcerans PE 170 70 PPE 105 46 ESX 24 13 Ext-ESX 5 3 Pseudos 0 109 Favors extracellular growth?

  35. 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

  36. 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?

  37. 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 Collaborators B.G. Barrell S.V. Gordon R.G. Hewinson J. Parkhill L. Ramakrishnan P.L.C. Small NIH NIAID ILEP AFRF

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