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

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.