Protease and transmembrane domain of the type VII secretion mycosin protease determine system-specific functioning in mycobacteria

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their highly hydrophobic diderm cell envelope. Pathogenic mycobacteria, such as Mycobacterium tuberculosis and Mycobacterium marinum, have up to five of these systems, named ESX-1 to -5. Most of these systems contain a set of five conserved membrane components, of which the four Ecc proteins form the core membrane-embedded secretion complex. The fifth conserved membrane protein, the mycosin protease (MycP), is not part of the core complex, but is essential for secretion, as it stabilizes this membrane complex. Here, we investigated which MycP domain is required for this stabilization by producing hybrid constructs between MycP1 and MycP5 in M. marinum and analyzed their effect on ESX-1 and ESX-5 secretion. We found that both the protease and transmembrane (TM) domain are required for the ESX system-specific function of mycosins. In addition, we observed that the TM domain strongly affects MycP protein levels. We also show that the extended loops 1 and 2 in the protease domain are probably primarily involved in MycP stability, whereas loop 3 and the MycP5-specific loop 5 are dispensable. The atypical propeptide, or N-terminal extension, is required only for MycP stability. Finally, we show that the protease domain of MycPP1, encoded by the esx-P1 locus on the pRAW plasmid, is functionally redundant to the protease domain of MycP5 These results provide the first insight into the regions of mycosins involved in the interaction with and the stabilization of their respective ESX complexes.