The ESX-1 substrate PPE68 has a key function in the ESX-1 mediated secretion inMycobacterium marinum

AbstractMycobacteria use specialized type VII secretion systems (T7SSs) to secrete proteins across their diderm cell envelope. One of the T7SS subtypes, named ESX-1, is a major virulence determinant in pathogenic species such asMycobacterium tuberculosisand the fish pathogenMycobacterium marinum.ESX-1 secretes a variety of substrates, called Esx, PE, PPE and Esp proteins, at least some of which as folded heterodimers. Investigations into the functions of these substrates is problematic, because of the intricate network of co-dependent secretion between several ESX-1 substrates. Here, we describe that the ESX-1 substrate PPE68 is essential for secretion of the highly immunogenic substrate EsxA and EspE via the ESX-1 system inM. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 ofM. marinumandM. tuberculosisis present in a cytosolic complex with its PE partner and the EspG1chaperone. Interfering with the binding of EspG1to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast,esxAis not required for the secretion of PPE68, revealing hierarchy in co-dependent secretion. Remarkably, the final ten residues of PPE68, a negatively charged domain, seem essential for EspE secretion, but not for the secretion of EsxA and PPE68 itself. This indicates that distinctive domains of PPE68 are involved in secretion of the different ESX-1 substrates. Based on these findings, we propose a mechanistic model for the central role of PPE68 in ESX-1 mediated secretion and substrate co-dependence.ImportanceMycobacterium tuberculosisuse type VII secretion systems (T7SSs) to secrete proteins across its uniquely hydrophobic diderm cell envelope. A T7SS subtype, called ESX-1, is one of its most important virulence determinants by mediating intracellular survival through phagosomal rupture and subsequent translocation of the mycobacterium to the host cytosol. Identifying the ESX-1 substrate that is responsible for this process is problematic because of the intricate network of co-dependent secretion between ESX-1 substrates. Here, we provide mechanistic insight into the central role of the ESX-1 substrate PPE68 for the secretion of ESX-1 substrates, using the model organismMycobacterium marinum. Unravelling the mechanism of co-dependent secretion will aid the functional understanding of T7SSs and will allow the analysis of the individual roles of ESX-1 substrates in the virulence caused by this significant human pathogen.