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

Abstract

Mycobacteria 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 as Mycobacterium tuberculosis and the fish pathogen Mycobacterium 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 in M. marinum. While secreted PPE68 is processed on the cell surface, the majority of cell-associated PPE68 of M. marinum and M. tuberculosis is present in a cytosolic complex with its PE partner and the EspG1 chaperone. Interfering with the binding of EspG1 to PPE68 blocked its export and the secretion of EsxA and EspE. In contrast, esxA is 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.

Reconstitution of a minimal ESX-5 type VII secretion system suggests a role for PPE proteins in outer membrane transport of proteins

“…PE/PPE proteins require Esx heterodimers to be secreted. This is in line with a recent observation for ESX-1 in Mycobacterium marinum , where secretion of EsxA is dependent on the co-secretion of PPE68 that is encoded by the same gene cluster (Damen et al , 2022). Notably, PPE68 was observed to be degraded on the cell surface upon export.…”

“…In the first model, the roles of the two esx-5 locus encoded PPE proteins in secretion are different, considering the differences in localization and membrane association. Here, the first PPE protein could play a role in activation of the inner membrane complex and co-secretion together with the EsxM/N heterodimer, similar as for the ESX-1 dependent Esx and PE/PPE substrates (Damen et al, 2022) , while the second PPE protein could form the channel for protein translocation over the mycomembrane. In the second model, the two PPE proteins form the mycomembrane channel together, with the second PPE protein creating the integral part and the first PPE being the peripheral portion of the complex.…”