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, includingMycobacterium tuberculosisand the fish pathogenMycobacterium marinum. We have previously shown that theM. tuberculosisESX-5 system is unable to fully complement secretion in anM. marinum esx-5mutant, suggesting species specificity in secretion. In this study, we elaborated on this observation and established that the membrane ATPase EccC(5), possessing four (putative) nucleotide-binding domains (NBDs), is responsible for this. By creatingM. marinum-M. tuberculosisEccC(5)chimeras, we observed both inM. marinumand inM. tuberculosisthat secretion specificity of PE_PGRS proteins depends on the presence of the cognate linker 2 domain of EccC(5). This region connects NBD1 and NBD2 of EccC(5)and is responsible for keeping NBD1 in an inhibited state. Notably, the ESX-5 substrate EsxN, predicted to bind to NBD3 on EccC(5), 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 EccC(5).