Species‐specific Secretion of ESX‐5 Type VII Substrates is Determined by the Linker 2 of EccC5

Mycobacteria use type VII secretion systems (T7SSs) to translocate a wide range of proteins across their diderm cell envelope. These systems, also called ESX systems, are crucial for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum. We have previously shown that the M. tuberculosis ESX‐5 system is unable to fully complement secretion in an M. marinum esx‐5 mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC5, possessing four (putative) nucleotide‐binding domains (NBDs), is responsible for this. By creating M. marinum‐M. tuberculosis EccC5 chimeras, we observed both in M. marinum and in M. tuberculosis that secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC5. This region connects NBD1 and NBD2 of EccC5 and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX‐5 substrate EsxN, predicted to bind to NBD3 on EccC5, showed a distinct secretion profile. These results indicate that linker 2 is involved in species‐specific substrate recognition and might therefore be an additional substrate recognition site of EccC5.