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

Area of Science:

• Microbiology
• Molecular Biology
• Protein Biochemistry

Background:

• Mycosin protease (MycP) is vital for type VII secretion (T7S) systems in Actinobacteria, particularly the ESX systems in mycobacteria.
• MycP's essential role in secretion is linked to stabilizing the ESX membrane complex, but its mechanism and interaction partners remain unclear.
• MycP is not a stable component of the ESX complex, posing a challenge to understanding its function.

Purpose of the Study:

• To investigate the interaction between Mycosin protease (MycP) and EccB within the ESX-5 secretion system.
• To determine if a chimeric fusion protein of EccB and MycP can restore ESX-5 secretion.
• To elucidate the role of MycP in the ESX complex stability and secretion mechanism.

Main Methods:

• Constructed a chimeric gene encoding an EccB5-MycP5 fusion protein.
• Utilized knockout strains in Mycobacterium marinum and Mycobacterium smegmatis to test the functional complementation of the fusion protein.
• Performed protein pulldown assays using EccC5 as bait to analyze the ESX-5 complex composition.

Main Results:

• The EccB5-MycP5 fusion protein successfully restored ESX-5 secretion in both eccB5 and mycP5 knockout strains.
• Reconstitution of the Mycobacterium xenopi esx-5 locus with the chimeric gene in Mycobacterium smegmatis also restored ESX-5 secretion.
• Protein pulldown assays confirmed that the EccB5-MycP5 fusion protein is a stable component of the ESX-5 complex and specifically copurified with EccC5.

Conclusions:

• MycP5 functions in close proximity to EccB5, suggesting EccB5 is the direct interaction partner of MycP5.
• The fusion strategy demonstrates that MycP5's essential secretion function can be mediated through interaction with EccB5.
• This study provides key insights into the structural organization and functional mechanism of the ESX-5 secretion system.