Species-specific secretion of ESX-5 type VII substrates is determined by the linker 2 of EccC ₅

Abstract Type VII secretion systems (T7SSs) are used by mycobacteria to translocate a wide range of effector proteins across their diderm cell envelope. These systems, also known as ESX systems, have crucial roles for the viability and/or virulence of mycobacterial pathogens, including Mycobacterium tuberculosis and the fish pathogen Mycobacterium marinum . We previously observed species-specificity in the secretion of the PE_PGRS proteins by the ESX-5 system [1], in that the M. tuberculosis ESX-5 system was unable to fully complement an M. marinum esx-5 mutant. In this study, we established that the responsible factor for this is the central membrane ATPase EccC 5 , which has three nucleotide binding domains (NBDs). By creating chimeric M. marinum / M. tuberculosis EccC 5 constructs, we observed that PE_PGRS secretion is mediated only in the presence of an EccC 5 containing the cognate linker 2, irrespective of the origin of the EccC 5 backbone. This region is responsible for linking the first two NBDs and for keeping the first NBD in an inhibited state. Notably, this region is disordered in a EccC crystal structure and is particularly extended in EccC proteins of the different ESX-5 systems. These results indicate that this region is involved in species-specific substrate recognition and might therefore be an additional substrate recognition site of EccC 5 .