Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis

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Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported by the different type VII secretion systems1,2. The most recently evolved type VII secretion system, ESX-5, secretes dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the amino terminus3,4. However, the role of these proteins remains largely elusive1. Here, we show that mutations of ppe38 completely block the secretion of two large subsets of ESX-5 substrates, that is, PPE-MPTR and PE_PGRS, together comprising >80 proteins. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have such a mutation and a concomitant loss of secretion5. Restoration of PPE38-dependent secretion partially reverted the hypervirulence phenotype of a Beijing strain, and deletion of ppe38 in moderately virulent M. tuberculosis increased virulence. This indicates that these ESX-5 substrates have an important role in virulence attenuation. Phylogenetic analysis revealed that deletion of ppe38 occurred at the branching point of the ‘modern’ Beijing sublineage and is shared by Beijing outbreak strains worldwide, suggesting that this deletion may have contributed to their success and global distribution6,7.

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Fig. 1: PPE38 is required for the secretion of PE_PGRS proteins and is itself secreted by M. marinum.
Fig. 2: PPE38/71 is required for the secretion of PE_PGRS proteins in M. tuberculosis.
Fig. 3: Loss of PPE38/71 increases virulence of M. tuberculosis in a mouse model.
Fig. 4: Phylogenetic analysis reveals ppe38 mutations are widespread in ‘modern’ Beijing strains.


  1. 1.

    Gröschel, M. I., Sayes, F., Simeone, R., Majlessi, L. & Brosch, R. ESX secretion systems: mycobacterial evolution to counter host immunity. Nat. Rev. Microbiol. 14, 677–691 (2016).

  2. 2.

    Ates, L. S., Houben, E. N. G. & Bitter, W. in Virulence Mechanisms of Bacterial Pathogens 5th edn (eds Kuvda, I. T. et al.) 357–384 (American Society of Microbiology, Washington DC, 2016).

  3. 3.

    Ates, L. S. et al. Essential role of the ESX-5 secretion system in outer membrane permeability of pathogenic mycobacteria. PLoS Genet. 11, e1005190 (2015).

  4. 4.

    Gey van Pittius, N. C. et al. Evolution and expansion of the Mycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol. Biol. 6, 95 (2006).

  5. 5.

    McEvoy, C. R. E., van Helden, P. D., Warren, R. M. & Gey van Pittius, N. C. Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region. BMC Evol. Biol. 9, 237 (2009).

  6. 6.

    Merker, M. et al. Evolutionary history and global spread of the Mycobacterium tuberculosis Beijing lineage. Nat. Genet. 47, 242–249 (2015).

  7. 7.

    Hanekom, M. et al. Mycobacterium tuberculosis Beijing genotype: a template for success. Tuberculosis (Edinb.) 91, 510–523 (2011).

  8. 8.

    WHO Global Tuberculosis Report 2015 (World Health Organization, 2015);

  9. 9.

    Aguilar, D. et al. Mycobacterium tuberculosis strains with the Beijing genotype demonstrate variability in virulence associated with transmission. Tuberculosis (Edinb.) 90, 319–325 (2010).

  10. 10.

    Reed, M. B. et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431, 84–87 (2004).

  11. 11.

    Weerdenburg, E. M. et al. ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish. Cell. Microbiol. 14, 728–739 (2012).

  12. 12.

    Abdallah, A. M. et al. PPE and PE_PGRS proteins of Mycobacterium marinum are transported via the type VII secretion system ESX-5. Mol. Microbiol. 73, 329–340 (2009).

  13. 13.

    Ates, L. S. et al. The ESX-5 system of pathogenic mycobacteria is involved in capsule integrity and virulence through its substrate PPE10. PLoS Pathog. 12, e1005696 (2016).

  14. 14.

    Saini, N. K. et al. Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47. Nat. Microbiol. 1, 16133 (2016).

  15. 15.

    Dong, D. et al. PPE38 modulates the innate immune response and is required for Mycobacterium marinum virulence. Infect. Immun. 80, 43–54 (2012).

  16. 16.

    Cole, S. T. et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544 (1998).

  17. 17.

    Bitter, W. et al. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog. 5, e1000507 (2009).

  18. 18.

    Hanekom, M. et al. A recently evolved sublineage of the Mycobacterium tuberculosis Beijing strain family is associated with an increased ability to spread and cause disease. J. Clin. Microbiol. 45, 1483–1490 (2007).

  19. 19.

    Strong, M. et al. Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 103, 8060–8065 (2006).

  20. 20.

    Abdallah, A. M. et al. A specific secretion system mediates PPE41 transport in pathogenic mycobacteria. Mol. Microbiol. 62, 667–679 (2006).

  21. 21.

    Korotkova, N. et al. Structure of the Mycobacterium tuberculosis type VII secretion system chaperone EspG 5 in complex with PE25–PPE41 dimer. Mol. Microbiol. 94, 367–382 (2014).

  22. 22.

    Shah, S., Cannon, J. R., Fenselau, C. & Briken, V. A duplicated ESAT-6 region of ESX-5 is involved in protein export and virulence of mycobacteria. Infect. Immun. 83, 4349–4361 (2015).

  23. 23.

    Bottai, D. et al. Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation. Mol. Microbiol. 83, 1195–1209 (2012).

  24. 24.

    Sayes, F. et al. Strong immunogenicity and cross-reactivity of Mycobacterium tuberculosis ESX-5 type VII secretion: encoded PE–PPE proteins predicts vaccine potential. Cell Host Microbe 11, 352–363 (2012).

  25. 25.

    Reed, M. B., Gagneux, S., Deriemer, K., Small, P. M. & Barry, C. E. The W-Beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated. J. Bacteriol. 189, 2583–2589 (2007).

  26. 26.

    Sinsimer, D. et al. The phenolic glycolipid of Mycobacterium tuberculosis differentially modulates the early host cytokine response but does not in itself confer hypervirulence. Infect. Immun. 76, 3027–3036 (2008).

  27. 27.

    Brites, D. & Gagneux, S. Old and new selective pressures on Mycobacterium tuberculosis. Infect. Genet. Evol. 12, 678–685 (2012).

  28. 28.

    Cole, S. T. et al. Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011 (2001).

  29. 29.

    Bardarov, S. et al. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 94, 10961–10966 (1997).

  30. 30.

    Vizcaíno, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, D447–D456 (2016).

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We thank N. C. Gey van Pittius, B. Appelmelk, J. Luirink and A. van der Sar for useful discussions and help with data interpretation. We also thank M. Sparrius, V. van Winden, R. Simeone and M. Kok for technical assistance. Furthermore we thank members of the Pathogen Genomics group and the Bioscience Core laboratory in King Abdullah University of Science and Technology (KAUST) for generating the sequencing data on the M. tuberculosis isolates described in the study. We also thank T. Phan for LC-MS/MS data analysis. E.N.G.H. was funded by a VIDI grant from the Netherlands Organization of Scientific Research. R.H.-P. was funded by grant CONACyT contract FC 2015-/115 and IMMUNOCANEI grant 253053. A.P. is funded by a faculty baseline funding (BAS/1/1020-01-01) by KAUST. L.S.A., F.L.C. and R.B. acknowledge support by grants ANR-14-JAMR-001-02 and ANR-10-LABX-62-IBEID and the European Union’s Horizon 2020 Research and Innovation Program grant 643381. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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L.S.A., R.U., S.R.P., A.D., K.v.d.K., A.D.v.d.W., F.L.C., B.M.-C., J.B.-P., D.M.-E. and C.G. performed the experiments. L.S.A., E.N.G.H., A.P., A.D., J.B.-P., R.H.-P., R.B. and W.B. contributed to the manuscript. L.S.A., A.D., S.R.P., R.M.W., R.H.-P. and W.B. performed the data analysis. C.R.J., A.P., J.B.-P., R.H.-P., R.M.W. and R.B. contributed reagents and/or facilities.

Correspondence to Louis S. Ates or Wilbert Bitter.

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Supplementary Information

Supplementary Tables 1–8, Supplementary Figures 1–12, Supplementary Discussion, Supplementary Methods, Supplementary References.

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Supplementary Table 3

Small insertions and deletions (indels) identified in strains SAWC_1945, SAWC_2135 and SAWC_2701.

Supplementary Table 4

Single-nucleotide polymorphisms identified in strains SAWC_1945, SAWC_2135.

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