Article

pks5-recombination-mediated surface remodelling in Mycobacterium tuberculosis emergence

  • Nature Microbiology 1, Article number: 15019 (2016)
  • doi:10.1038/nmicrobiol.2015.19
  • Download Citation
Received:
Accepted:
Published online:

Abstract

Mycobacterium tuberculosis is a major, globally spread, aerosol-transmitted human pathogen, thought to have evolved by clonal expansion from a Mycobacterium canettii-like progenitor. In contrast, extant M. canettii strains are rare, genetically diverse, and geographically restricted mycobacteria of only marginal epidemiological importance. Here, we show that the contrasting evolutionary success of these two groups is linked to loss of lipooligosaccharide biosynthesis and subsequent morphotype changes. Spontaneous smooth-to-rough M. canettii variants were found to be mutated in the polyketide-synthase-encoding pks5 locus and deficient in lipooligosaccharide synthesis, a phenotype restored by complementation. Importantly, these rough variants showed an altered host–pathogen interaction and increased virulence in cellular- and animal-infection models. In one variant, lipooligosaccharide deficiency occurred via homologous recombination between two pks5 genes and removal of the intervening acyltransferase-encoding gene. The resulting single pks5 configuration is similar to that fixed in M. tuberculosis, which is known to lack lipooligosaccharides. Our results suggest that pks5-recombination-mediated bacterial surface remodelling increased virulence, driving evolution from putative generalist mycobacteria towards professional pathogens of mammalian hosts.

  • Subscribe to Nature Microbiology for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

References

  1. 1.

    , , & Mycobacterial pathogenomics and evolution. Microbiol. Spectrum 2, MGM2-0025-2013 (2014).

  2. 2.

    , , , & Mycobacterium tuberculosis evolutionary pathogenesis and its putative impact on drug development. Future Microbiol. 9, 969–985 (2014).

  3. 3.

    et al. Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis. Nature Genet. 45, 172–179 (2013).

  4. 4.

    et al. A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa. Int. J. Syst. Bacteriol. 47, 1236–1245 (1997).

  5. 5.

    et al. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog. 1, e5 (2005).

  6. 6.

    et al. Clinical characteristics of the smooth tubercle bacilli ‘Mycobacterium canettii’ infection suggest the existence of an environmental reservoir. Clin. Microbiol. Infect. 17, 1013–1019 (2011).

  7. 7.

    et al. Progenitor ‘Mycobacterium canettii’ clone responsible for lymph node tuberculosis epidemic, Djibouti. Emerg. Infect. Dis. 20, 21–28 (2014).

  8. 8.

    et al. Correlation of virulence, lung pathology, bacterial load and delayed type hypersensitivity responses after infection with different Mycobacterium tuberculosis genotypes in a BALB/c mouse model. Clin. Exp. Immunol. 137, 460–468 (2004).

  9. 9.

    et al. A glimpse into the past and predictions for the future: the molecular evolution of the tuberculosis agent. Mol. Microbiol. 93, 835–852 (2014).

  10. 10.

    et al. Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids. Proc. Natl Acad. Sci. USA 111, 13264–13271 (2014).

  11. 11.

    , & Change in colony morphology influences the virulence as well as the biochemical properties of the Mycobacterium avium complex. Microb. Pathog. 25, 203–214 (1998).

  12. 12.

    et al. Hypervirulence of a rough variant of the Mycobacterium abscessus type strain. Infect. Immun. 75, 1055–1058 (2007).

  13. 13.

    & Chemical basis of rough and smooth variation in mycobacteria. J. Bacteriol. 171, 3465–3470 (1989).

  14. 14.

    et al. Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum. J. Biol. Chem. 287, 20417–20429 (2012).

  15. 15.

    & Isolation in high frequency of rough variants of Mycobacterium intracellulare lacking C-mycoside glycopeptidolipid antigens. J. Bacteriol. 150, 381–384 (1982).

  16. 16.

    et al. Spontaneous reversion of Mycobacterium abscessus from a smooth to a rough morphotype is associated with reduced expression of glycopeptidolipid and reacquisition of an invasive phenotype. Microbiology 152, 1581–1590 (2006).

  17. 17.

    et al. Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus. Mol. Microbiol. 90, 612–629 (2013).

  18. 18.

    et al. LosA, a key glycosyltransferase involved in the biosynthesis of a novel family of glycosylated acyltrehalose lipooligosaccharides from Mycobacterium marinum. J. Biol. Chem. 280, 42124–42133 (2005).

  19. 19.

    et al. Identification of the lipooligosaccharide biosynthetic gene cluster from Mycobacterium marinum. Mol. Microbiol. 63, 1345–1359 (2007).

  20. 20.

    , , & Lack of correlation between colony morphology and lipooligosaccharide content in the Mycobacterium tuberculosis complex. J. Gen. Microbiol. 138, 1535–1541 (1992).

  21. 21.

    et al. Identification of the polyketide synthase involved in the biosynthesis of the surface-exposed lipooligosaccharides in mycobacteria. J. Bacteriol. 191, 2613–2621 (2009).

  22. 22.

    et al. MKAN27435 is required for the biosynthesis of higher subclasses of lipooligosaccharides in Mycobacterium kansasii. PLoS ONE 10, e0122804 (2015).

  23. 23.

    et al. in Tuberculosis—Expanding Knowledge (ed. Ribon, W.) Ch. 7 (InTech, 2015).

  24. 24.

    et al. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res. 18, 729–741 (2008).

  25. 25.

    et al. Insights on the emergence of Mycobacterium tuberculosis from the analysis of Mycobacterium kansasii. Genome Biol. Evol. 7, 856–870 (2015).

  26. 26.

    Biosynthesis of mycobacterial lipids by polyketide synthases and beyond. Crit. Rev. Biochem. Mol. Biol. 49, 179–211 (2014).

  27. 27.

    et al. Virulence attenuation of two Mas-like polyketide synthase mutants of Mycobacterium tuberculosis. Microbiology 149, 1837–1847 (2003).

  28. 28.

    , & Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis. Tuber. Lung Dis. 79, 171–180 (1999).

  29. 29.

    , & Novel type-specific lipooligosaccharides from Mycobacterium tuberculosis. Biochemistry 30, 378–388 (1991).

  30. 30.

    , , , & The cell envelope glycoconjugates of Mycobacterium tuberculosis. Crit. Rev. Biochem. Mol. Biol. 49, 361–399 (2014).

  31. 31.

    , , , & Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5. Proc. Natl Acad. Sci. USA 101, 4608–4613 (2004).

  32. 32.

    , , , & Two polyketide-synthase-associated acyltransferases are required for sulfolipid biosynthesis in Mycobacterium tuberculosis. Microbiology 153, 513–520 (2007).

  33. 33.

    et al. Increased protective efficacy of recombinant BCG strains expressing virulence-neutral proteins of the ESX-1 secretion system. Vaccine 33, 2710–2718 (2015).

  34. 34.

    et al. Mycobacterium abscessus glycopeptidolipids mask underlying cell wall phosphatidyl-myo-inositol mannosides blocking induction of human macrophage TNF-alpha by preventing interaction with TLR2. J. Immunol. 183, 1997–2007 (2009).

  35. 35.

    et al. Overexpression of proinflammatory TLR-2-signalling lipoproteins in hypervirulent mycobacterial variants. Cell Microbiol. 13, 692–704 (2011).

  36. 36.

    , & The onset of adaptive immunity in the mouse model of tuberculosis and the factors that compromise its expression. Immunol. Rev. 264, 46–59 (2015).

  37. 37.

    Insights from genomic comparisons of genetically monomorphic bacterial pathogens. Phil. Trans. R. Soc. B 367, 860–867 (2012).

  38. 38.

    et al. A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc. Natl Acad. Sci. USA 101, 314–319 (2004).

  39. 39.

    et al. Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene. J. Biol. Chem. 277, 38148–38158 (2002).

  40. 40.

    et al. Mycobacterium tuberculosis pks12 produces a novel polyketide presented by CD1c to T cells. J. Exp. Med. 200, 1559–1569 (2004).

  41. 41.

    et al. Fatty acyl chains of Mycobacterium marinum lipooligosaccharides: structure, localization and acylation by PapA4 (MMAR_2343) protein. J. Biol. Chem. 286, 33678–33688 (2011).

  42. 42.

    et al. Increased phagocytosis of Mycobacterium marinum mutants defective in lipooligosaccharide production: a structure–activity relationship study. J. Biol. Chem. 289, 215–228 (2014).

  43. 43.

    , , & A genetic mechanism for deletion of the ser2 gene cluster and formation of rough morphological variants of Mycobacterium avium. J. Bacteriol. 182, 6177–6182 (2000).

  44. 44.

    et al. Identification of the surface-exposed lipids on the cell envelopes of Mycobacterium tuberculosis and other mycobacterial species. J. Bacteriol. 178, 456–461 (1996).

  45. 45.

    et al. Interaction of pattern recognition receptors with Mycobacterium tuberculosis. J. Clin. Immunol. 35, 1–10 (2015).

  46. 46.

    et al. Mycobacteria manipulate macrophage recruitment through coordinated use of membrane lipids. Nature 505, 218–222 (2014).

  47. 47.

    , , & Vitamin B12 metabolism in Mycobacterium tuberculosis. Future Microbiol. 8, 1405–1418 (2013).

  48. 48.

    , & Phylogenetic analysis of vitamin B12-related metabolism in Mycobacterium tuberculosis. Front. Mol. Biosci. 2, 6 (2015).

  49. 49.

    et al. An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity. Proc. Natl Acad. Sci. USA 111, 6750–6755 (2014).

  50. 50.

    et al. The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli. J. Biol. Chem. 274, 35969–35974.

  51. 51.

    , , , & SHRiMP2: sensitive yet practical SHort Read Mapping. Bioinformatics 27, 1011–1012 (2011).

  52. 52.

    et al. Using Tablet for visual exploration of second-generation sequencing data. Brief Bioinform. 14, 193–202 (2013).

  53. 53.

    & Accurate whole-genome sequencing-based epidemiological surveillance of Mycobacterium tuberculosis. Methods Microbiol 42, 359–394 (2015).

  54. 54.

    & Application of phylogenetic networks in evolutionary studies. Mol. Biol. Evol. 23, 254–267 (2006).

  55. 55.

    et al. Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast 17, 111–123 (2000).

  56. 56.

    et al. Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect. Immun. 66, 2221–2229 (1998).

  57. 57.

    , , , & Comparative genomic analysis of mycobacteriophage Tweety: evolutionary insights and construction of compatible site-specific integration vectors for mycobacteria. Microbiology 153, 2711–2723 (2007).

  58. 58.

    et al. Xer site-specific recombination, an efficient tool to introduce unmarked deletions into mycobacteria. Appl. Environ. Microbiol. 76, 5312–5316 (2010).

  59. 59.

    et al. Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure. Mol. Microbiol. 52, 725–733 (2004).

  60. 60.

    et al. Influence of ESAT-6 secretion system 1 (RD1) of Mycobacterium tuberculosis on the interaction between mycobacteria and the host immune system. J. Immunol. 174, 3570–3579 (2005).

Download references

Acknowledgements

The authors thank T. Seemann for initial help with NeighborNet analysis, and H. Pouseele for help with mapping and SNP analysis. The authors also thank I. Rosenkrands and G. Delogu for providing polyclonal anti-SigA antibodies and vector pMV10-25, respectively, and K. Sébastien for expert assistance in animal care in the biosafety-A3 facilities. The authors acknowledge support from a European Community grant (no. 260872), the EU-EFPIA Innovative Medicines Initiative (grant no. 115337), the Agence National de Recherche (ANR-14-JAMR-001-02) and the Fondation pour la Recherche Médicale FRM (DEQ20090515399 and DEQ20130326471). High-throughput sequencing was performed on the Genomics Platform, a member of the ‘France Génomique’ consortium (ANR10-INBS-09-08). R.B. is a member of the LabEx consortium IBEID at the Institut Pasteur. F.L.-C. was supported by the French Region Ile-de-France (Domaine d'Intérêt Majeur Maladies Infectieuses et Emergentes) PhD programme. E.C.B. was supported by a stipend from the Pasteur–Paris University (PPU) International PhD programme and the Institut Carnot Pasteur Maladies Infectieuses.

Author information

Affiliations

  1. Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, Paris 75015, France

    • Eva C. Boritsch
    • , Wafa Frigui
    • , Alessandro Cascioferro
    • , Alexandre Pawlik
    • , Fabien Le Chevalier
    • , Mickael Orgeur
    • , Laleh Majlessi
    •  & Roland Brosch
  2. CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse 31000, France

    • Wladimir Malaga
    • , Gilles Etienne
    • , Françoise Laval
    • , Mamadou Daffé
    •  & Christophe Guilhot
  3. Université de Toulouse, UPS, IPBS, Toulouse 31000, France

    • Wladimir Malaga
    • , Gilles Etienne
    • , Françoise Laval
    • , Mamadou Daffé
    •  & Christophe Guilhot
  4. University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France

    • Fabien Le Chevalier
  5. Institut Pasteur, PF1-Plate-Forme Génomique, Paris, France

    • Laurence Ma
    •  & Christiane Bouchier
  6. Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia

    • Timothy P. Stinear
  7. Inserm U1019, CNRS UMR8204, Université de Lille, Institut Pasteur de Lille, Center for Infection and Immunity, Lille 59000, France

    • Philip Supply

Authors

  1. Search for Eva C. Boritsch in:

  2. Search for Wafa Frigui in:

  3. Search for Alessandro Cascioferro in:

  4. Search for Wladimir Malaga in:

  5. Search for Gilles Etienne in:

  6. Search for Françoise Laval in:

  7. Search for Alexandre Pawlik in:

  8. Search for Fabien Le Chevalier in:

  9. Search for Mickael Orgeur in:

  10. Search for Laurence Ma in:

  11. Search for Christiane Bouchier in:

  12. Search for Timothy P. Stinear in:

  13. Search for Philip Supply in:

  14. Search for Laleh Majlessi in:

  15. Search for Mamadou Daffé in:

  16. Search for Christophe Guilhot in:

  17. Search for Roland Brosch in:

Contributions

E.C.B., C.G., L. Majlessi and R.B. designed the study. E.C.B., W.F., F.L.C. and A.P. performed mycobacterial phenotypic assays and/or infection experiments. E.C.B., A.C. and R.B. established genetic constructs. W.M., G.E., F.L., M.D. and C.G. generated and/or analysed mycobacterial lipid and lipooligosaccharide profiles. E.C.B., L.Ma, C.B., M.O., T.P.S. and P.S. generated and/or analysed sequence data. E.C.B and L.Majlessi conducted and analysed immune assays. E.C.B., T.P.S., P.S., C.G. and R.B. wrote the manuscript, with comments from all authors.

Competing interests

P.S. is a consultant for Genoscreen. All other authors declare no competing financial interests.

Corresponding authors

Correspondence to Christophe Guilhot or Roland Brosch.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1–13, Tables 1–5, Note, References and raw data (gels, blots and TLCs).