Rerouting of an Esx substrate pair from the ESX-1 type VII secretion system to ESX-5 by modifying a PE/PPE substrate pair

Type VII secretion systems (T7SSs) secrete a wide range of extracellular proteins that play important roles in bacterial viability and in host-pathogen interactions of pathogenic mycobacteria. There are five subtypes of mycobacterial T7SSs, called ESX-1 to ESX-5, and four classes of T7SS substrates, namely the Esx, PE, PPE and Esp proteins. At least some of these substrates are secreted as heterodimers. The ESX systems mediate the secretion of specific members of the Esx, PE and PPE proteins, raising the question how these substrates are recognized in a system-specific fashion. PE/PPE heterodimers interact with their cognate EspG chaperones, which recently has been shown to determine their designated secretion pathway. Both structural and pulldown analysis suggest that EspG is unable to interact with Esx proteins and therefore the determining factor for system-specificity of these substrates remains unknown. In this study, we have investigated the secretion specificity of the ESX-1 substrate pair EsxB\_1/EsxA\_1 (MMAR\_0187/MMAR \_0188) in Mycobacterium marinum . While this substrate pair was hardly secreted when ectopically expressed, secretion was observed when EsxB\_1/EsxA\_1 was co-expressed together with PE35/PPE68\_1 (MMAR\_0185/MMAR\_0186), which are encoded by the same operon. Surprisingly, co-expressing EsxB\_1/EsxA\_1 with a modified PE35/PPE68\_1 version that carried the EspG5 chaperone binding domain, previously shown to redirect this substrate pair to the ESX-5 system, also resulted in co-secretion of EsxB\_1/EsxA\_1 via ESX-5. Our data suggest a secretion model in which PE35/PPE68\_1 is a determinant factor for the system-specific secretion of EsxB\_1/EsxA_1. * T7SS : Type VII secretion system; NBD : nucleotide binding domain; PBS : phosphate buffered saline; TCA : trichloroacetic acid