Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.

Department of Cell Biology and Histology Academic Medical Center University of Amsterdam Amsterdam the Netherlands

Department of Medical Microbiology and Infection Prevention VU University Medical Center Amsterdam the Netherlands

Department of Medical Microbiology and Infection Prevention VU University Medical Center Amsterdam the Netherlands Department of Molecular Microbiology VU University Amsterdam the Netherlands

Department of Molecular Cell Biology and Immunology VU University Medical Center Amsterdam the Netherlands

Department of Molecular Microbiology VU University Amsterdam the Netherlands

Department of Physiology and Cardiology VU University Medical Center Amsterdam the Netherlands

Eyen SE Prague Czech Republic

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$a Ates, Louis S $u Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands. $7 gn_A_00009731

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$a The ESX-5 System of Pathogenic Mycobacteria Is Involved In Capsule Integrity and Virulence through Its Substrate PPE10 / $c LS. Ates, AD. van der Woude, J. Bestebroer, G. van Stempvoort, RJ. Musters, JJ. Garcia-Vallejo, DI. Picavet, Rv. Weerd, M. Maletta, CP. Kuijl, NN. van der Wel, W. Bitter,

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$a Mycobacteria produce a capsule layer, which consists of glycan-like polysaccharides and a number of specific proteins. In this study, we show that, in slow-growing mycobacteria, the type VII secretion system ESX-5 plays a major role in the integrity and stability of the capsule. We have identified PPE10 as the ESX-5 substrate responsible for this effect. Mutants in esx-5 and ppe10 both have impaired capsule integrity as well as reduced surface hydrophobicity. Electron microscopy, immunoblot and flow cytometry analyses demonstrated reduced amounts of surface localized proteins and glycolipids, and morphological differences in the capsular layer. Since capsular proteins secreted by the ESX-1 system are important virulence factors, we tested the effect of the mutations that cause capsular defects on virulence mechanisms. Both esx-5 and ppe10 mutants of Mycobacterium marinum were shown to be impaired in ESX-1-dependent hemolysis. In agreement with this, the ppe10 and esx5 mutants showed reduced recruitment of ubiquitin in early macrophage infection and intermediate attenuation in zebrafish embryos. These results provide a pivotal role for the ESX-5 secretion system and its substrate PPE10, in the capsular integrity of pathogenic mycobacteria. These findings open up new roads for research on the mycobacterial capsule and its role in virulence and immune modulation.

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$a van der Woude, Aniek D $u Department of Medical Microbiology and Infection Prevention, VU University Medical Center, Amsterdam, the Netherlands. Department of Molecular Microbiology, VU University, Amsterdam, the Netherlands.

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$a van Stempvoort, Gunny $u Department of Molecular Microbiology, VU University, Amsterdam, the Netherlands.

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