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

Nature Microbiology volume 3, pages 181–188 (2018)Cite this article

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Abstract

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.

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Acknowledgements

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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Authors and Affiliations

  1. Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, The Netherlands

    Louis S. Ates, Roy Ummels, Aniek D. van der Woude, Kim van der Kuij & Wilbert Bitter

  2. Unit for Integrated Mycobacterial Pathogenomics, Institut Pasteur, Paris, France

    Louis S. Ates, Fabien Le Chevalier & Roland Brosch

  3. DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, SAMRC Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Tygerberg, South Africa

    Anzaan Dippenaar & Robin M. Warren

  4. Department of Medical Oncology, OncoProteomics Laboratory, VU University Medical Center, Amsterdam, Netherlands

    Sander R. Piersma & Connie R. Jiménez

  5. Experimental Pathology Section, Department of Pathology, National Institute of Medical Sciences and Nutrition “Salvador Zubirán”, México City, Mexico

    Dulce Mata-Espinosa, Jorge Barrios-Payán, Brenda Marquina-Castillo, Carolina Guapillo & Rogelio Hernández-Pando

  6. Pathogen Genomics Laboratory, BESE Division, King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia

    Arnab Pain

  7. Section Molecular Microbiology, Amsterdam Institute of Molecules, Medicine & Systems, Vrije Universiteit, Amsterdam, The Netherlands

    Edith N. G. Houben & Wilbert Bitter

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  1. Louis S. Ates
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Contributions

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.

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Correspondence to Louis S. Ates or Wilbert Bitter.

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Supplementary information

Supplementary Information

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

Life Sciences Reporting Summary

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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Ates, L.S., Dippenaar, A., Ummels, R. et al. Mutations in ppe38 block PE_PGRS secretion and increase virulence of Mycobacterium tuberculosis. Nat Microbiol 3, 181–188 (2018). https://doi.org/10.1038/s41564-017-0090-6

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