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

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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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 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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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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