Abstract
Type VII secretion (T7S) systems are specialized machineries used by mycobacterial pathogens to transport important virulence factors across their highly hydrophobic cell envelope. There are up to five mycobacterial T7S systems, named ESX-1 to ESX-5, at least three of which specifically secrete a different subset of substrates. The T7S substrates or substrate complexes are defined by the general secretion motif YxxxD/E. However this motif does not determine system specificity. Here, we show that the substrate domain recognized by the EspG chaperone is the determinant factor for this specificity. We first show that the introduction of point mutations into the EspG1-binding domain of the ESX-1 substrate pair PE35/PPE68_1 affects their secretion. Subsequently, we demonstrate that replacing this domain by the EspG5-binding domain of the ESX-5 substrate PPE18 resulted in EspG5 dependence and exclusive rerouting to the ESX-5 system. This rerouting of PE35/PPE68_1 to the ESX-5 system had a negative effect on the secretion of endogenous ESX-5 substrates.
Highlights
Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient, but still one of the deadliest human pathogens[1]
Point mutations that changed this hydrophobic patch of the M. marinum ESX-5 substrates PPE18, which is secreted as a heterodimer together with PE31, and LipY abolished EspG5 binding and affected their secretion via the ESX-5 system[11]
The same C-terminal replacement did not have any effect on secretion of the PPE partner, when the PE35 hybrid was expressed in combination with PPE68_1 SWAP (Fig. 3A, lane 8). This combination showed a similar phenotype as PE35 WT/PPE68_1 SWAP, suggesting that exchanging the secretion motif did not affect the secretion capability of the PPE68_1 containing the EspG5 binding domain. These results show that PPE68_1 containing the EspG5-binding domain of PPE18 is efficiently secreted in the M. marinum WT strain, both with the presence of the ESX-1 or ESX-5 general secretion motif
Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis, is an ancient, but still one of the deadliest human pathogens[1]. One of the partners in this heterodimer contains a general secretion motif, i.e. YxxxD/E, directly after the second α-helix[9,11,12] This general secretion motif does not define system specificity[13], as the C-terminal 15 amino acids of an ESX-5 substrate containing this motif could be replaced by the homologous sequence of an ESX-1 substrate, and vice versa, without changing system specificity[13]. EspG5, the chaperone encoded by the esx-5 locus, interacts with PE/PPE proteins secreted via ESX-5, but not with an ESX-1 PE/PPE substrate pair[14]. EspG1, the chaperone encoded by the esx-1 locus, has been shown to bind only to its cognate substrate pair PE35/PPE68_1 and this component is essential for the solubility of the ESX-1 PE/PPE pair[14]. We have elucidated the role of the EspG-binding domain of PPE proteins in system specificity in M. marinum, by replacing the EspG1 binding domain of the ESX-1 substrates PE35/PP68_1 with the EspG5 binding domain of the ESX-5 substrate PPE18
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