Identification of a substrate domain that determines system specificity in mycobacterial type VII secretion systems

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.

New insights into the mycobacterial PE and PPE proteins provide a framework for future research

“…Structure of the EspG5‐PPE41‐PE25 complex (4KXR). The EspG chaperone (Gold) binds to the EspG‐binding domain of the PPE protein (Light blue), thereby conferring TypeVII secretion system specificity (Daleke et al ., ; Korotkova et al ., ; Phan et al ., ). The rest of the PPE‐protein interacts with its PE partner (Teal) via hydrophobic interactions.…”

“…Instead, they bind to cytosolic EspG chaperones and thereby confer system specificity to these substrates (Fig. ; Daleke et al , ; Ekiert and Cox, ; Korotkova et al , ; Phan et al , ; Phan and Houben, ). Intriguingly, exchanging the EspG interacting domain of the ESX‐1 secreted PPE68_1 with that of the ESX‐5 substrate PPE18 was sufficient to reroute this protein from ESX‐1 to ESX‐5 in M. marinum (Phan et al , ).…”

“…; Daleke et al , ; Ekiert and Cox, ; Korotkova et al , ; Phan et al , ; Phan and Houben, ). Intriguingly, exchanging the EspG interacting domain of the ESX‐1 secreted PPE68_1 with that of the ESX‐5 substrate PPE18 was sufficient to reroute this protein from ESX‐1 to ESX‐5 in M. marinum (Phan et al , ). Therefore, the sequence of the EspG‐binding domain of the PPE protein may be sufficient to predict system specificity of PPE proteins, although experimental validation of this hypothesis remains incomplete beyond the ESX‐1 and ESX‐5 systems (Ekiert and Cox, ; Korotkova et al , ; Chen et al , ; Phan and Houben, ).…”

“…These operonic clusters are likely to be the first pe and ppe genes duplicated from the esx genetic loci after introduction of the ESX‐5 system into the most‐recent common ancestor of the slow‐growing mycobacteria (Gey van Pittius et al , ; Fishbein et al , ; Dumas et al , ). The PE and PPE proteins encoded by such operons are thought to be exclusively secreted as PE and PPE heterodimers that may not be promiscuous to other secretion partners (Ekiert and Cox, ; Korotkova et al , ; Chen et al , ; Phan et al , ; Phan and Houben, ). A notable example of such operonic organization is the PE8‐PPE15‐EsxI‐EsxJ operon, which was shown to facilitate protein secretion of other TypeVII and non‐TypeVII substrates (Shah et al , ).…”

“…It is not yet known whether this is true only for PE_PGRS proteins, or also for other sublineage‐V PE‐proteins, that is, PPE38 required for secretion of the PGRS/MPTR domains specifically? Or, is it required for all sublineage‐V PE and PPE’s by for instance functioning as the partner in heterodimeric secretion complexes that can interact with the chaperone EspG 5 (Daleke et al , ; Phan et al , )? While this latter possibility was the major hypothesis upon identification of PPE38 as an essential factor for PE_PGRS secretion, it is hard to reconcile this hypothesis with the finding that PPE38 is also required for PPE‐MPTR secretion (Ates et al , ).…”