A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex

Vincent J.C. van Winden, Catalin M. Bunduc, Roy Ummels, Wilbert Bitter, Edith N.G. Houben

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

Original languageEnglish
JournalJournal of Molecular Biology
DOIs
Publication statusAccepted/In press - 1 Jan 2020

Cite this

@article{4a1abd84f478408f8e3e3f7ec9efbb9f,
title = "A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex",
abstract = "The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.",
keywords = "ESX, mycobacterium, MycP, protein secretion, type VII secretion system",
author = "{van Winden}, {Vincent J.C.} and Bunduc, {Catalin M.} and Roy Ummels and Wilbert Bitter and Houben, {Edith N.G.}",
year = "2020",
month = "1",
day = "1",
doi = "10.1016/j.jmb.2019.12.040",
language = "English",
journal = "Journal of Molecular Biology",
issn = "0022-2836",
publisher = "Academic Press Inc.",

}

A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex. / van Winden, Vincent J.C.; Bunduc, Catalin M.; Ummels, Roy; Bitter, Wilbert; Houben, Edith N.G.

In: Journal of Molecular Biology, 01.01.2020.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - A Chimeric EccB-MycP Fusion Protein is Functional and a Stable Component of the ESX-5 Type VII Secretion System Membrane Complex

AU - van Winden, Vincent J.C.

AU - Bunduc, Catalin M.

AU - Ummels, Roy

AU - Bitter, Wilbert

AU - Houben, Edith N.G.

PY - 2020/1/1

Y1 - 2020/1/1

N2 - The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

AB - The mycosin protease (MycP) is widely conserved in type VII secretion (T7S) systems throughout Actinobacteria. Within the T7S systems of mycobacteria, also known as the ESX systems, MycP is essential for secretion, which is probably linked to its stabilizing effect on the ESX membrane complex. However, it is unknown how this is mediated, as MycP is not a stable component of this complex. In this study, we set out to create a chimeric fusion protein of EccB5 and MycP5, based on a chimeric gene of eccB and mycP in the T7S locus of Bifidobacterium dentium. We show that this fusion protein is functional and capable of complementing ESX-5 secretion in both an eccB5 and a mycP5 knockout in Mycobacterium marinum. To study the ESX complex containing this fusion protein in more detail, we replaced the original eccB5 and mycP5 of the Mycobacterium xenopi esx-5 locus, reconstituted in Mycobacterium smegmatis, with the chimeric gene. The EccB5-MycP5 fusion construct also restored ESX-5 secretion under these double knockout conditions. Subsequent protein pulldowns on the central complex component EccC5 showed that under these conditions, the EccB5-MycP5 fusion was specifically copurified and a stable component of the ESX-5 complex. Based on our results, we can conclude that MycP5 carries out its essential function in secretion in close proximity to EccB5, indicating that EccB5 is the direct interaction partner of MycP5.

KW - ESX

KW - mycobacterium

KW - MycP

KW - protein secretion

KW - type VII secretion system

UR - http://www.scopus.com/inward/record.url?scp=85078767502&partnerID=8YFLogxK

U2 - 10.1016/j.jmb.2019.12.040

DO - 10.1016/j.jmb.2019.12.040

M3 - Article

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

SN - 0022-2836

ER -