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.


Introduction
Mycobacteria possess an unusual hydrophobic cell envelope that protects them from various stresses and contributes to the resilience of pathogenic mycobacteria during infection. Classified as high-GC Gram-positive bacteria, the cell envelope of mycobacteria consists of a standard cell membrane with a surrounding peptidoglycan layer. However, mycobacteria belong to a subgroup of high-GC Gram-positive bacteria that have acquired an extra hydrophobic layer of long-chain fatty acids, called mycolic acids. These specific lipids are covalently linked via an arabinogalactan layer to the peptidoglycan layer, forming a highly rigid and impermeable structure. Mycobacteria employ specialized machineries, called type VII secretion systems (T7SSs) to secrete proteins across their complex cell envelope (1,2). Mycobacterium tuberculosis possesses five of such T7SSs, named ESX-1 to ESX-5 (1,2), of which ESX-1, ESX-3 and ESX-5 have been functionally analysed (3)(4)(5)(6)(7)(8)(9)(10). Each of these systems plays a different role in the mycobacterial life cycle. For example, ESX-1 has a key role in virulence of pathogenic mycobacteria, as it mediates phagosomal rupture inside macrophages (11)(12)(13)(14) and the subsequent escape of M. tuberculosis from the phagolysosome (3-6, 15, 16). ESX-3 and ESX-5 are necessary for iron and fatty acid uptake, respectively, making them essential for bacterial viability (7)(8)(9)(10). Besides their roles in nutrient and metabolite acquisition, ESX-3 and ESX-5 have also been shown to be involved in immune modulation of the host (9,17,18).
The substrates that are secreted by these three ESX systems belong to distinctive protein families, i.e. Esx, PE, PPE and Esp proteins, most of them belonging to the so-called EsxAB clan protein superfamily (Pfam CL0352) (19). Some of these substrates have been shown to form heterodimers in the cytosol, i.e. two Esx proteins pair together and PE proteins pair with a PPE protein, and are thought to be secreted as (partially) folded dimers (13,(20)(21)(22)(23). Crystal structures have been solved for several heterodimeric substrates of different ESX systems, revealing highly conserved features, in which the interface of Esx heterodimers (24,25) as well as the interface of PE/PPE heterodimers, is formed by two pairs of alpha-helices oriented antiparallel to each other (21,22,26).
Interestingly, each ESX system secretes its own subset of Esx, PE and PPE substrates that are mostlikely responsible for the various roles of ESX systems in the bacterial life cycle. How these structurally similar proteins are specifically targeted to their corresponding ESX system still remains unclear. A conserved secretion signal (YxxxD/E) was identified, which is located directly after the helix-turn-helix domain of one partner protein of the Esx heterodimer and the PE partner of the PE/PPE heterodimer. This signal, although required for secretion, was shown to be exchangeable among PE substrates of different ESX systems without changing their initial secretion route (27,28).
Hence, this signal does not determine system-specific secretion of these T7SS substrates.
Structural analysis showed that PPE proteins have a relatively hydrophobic helical tip domain that extends from the characteristic four-helix bundle formed by the PE-PPE interface (21,22). This helical tip domain is recognized by a cytosolic chaperone, called EspG, in a system-specific manner PE/PPE determine system-specificity of Esx substrates 4 and this interaction is required for secretion of the PE/PPE pair (21,22,29). Subsequently, we could establish the redirection of the ESX-1 substrate pair PE35/PPE68_1 to the ESX -5 system by replacing   the EspG 1 chaperone binding domain with the equivalent domain of the ESX-5 substrate PPE18, suggesting this domain determines through which system these substrates are transported (30). The remaining question is how the Esx substrate pairs that lack this extended tip domain, are specifically recognized and targeted to their designated systems.
Here, we investigated the signals that determine the system-specificity of Esx substrates in M. marinum using the ESX-1 heterodimer EsxB_1/EsxA_1 as model substrates. Its encoding genes (MMAR_0185/MMAR_0186), which gene products we previously used as a model ESX-1 dependent PE/PPE heterodimer (29,30). We found that EsxB_1/EsxA_1 secretion via the ESX-1 system is severely enhanced by the co-expression and secretion of PE35/PPE68_1. Surprisingly, we were able to reroute the EsxB_1/EsxA_1 pair to the ESX-5 system by solely exchanging the EspG binding domain in PPE68_1, showing that the PE/PPE pair determines the system-specificity of this Esx pair.

EsxB_1/EsxA_1 require co-expression of PE35/PPE68_1 for efficient ESX-1 dependent secretion
To investigate how the system-specific secretion of Esx substrates is achieved, we investigated the secretion of EsxB_1/EsxA_1 in M. marinum. The corresponding coding genes (MMAR_0187/MMAR_0188) lie adjacent to the gene pair pe35/ppe68_1 (MMAR_0185/MMAR_0186) and are paralogues of the pe35-ppe68-esxB-esxA gene cluster located in the esx-1 locus (Fig. 1A). We introduced a shuttle plasmid containing esxB_1/esxA_1, expressed under the constitutive hsp60 promoter, in WT M. marinum (31). We also included WT M. marinum containing the previously analyzed pe35/ppe68_1 gene pair controlled by the same promoter on an integrative plasmid as an ESX-1 substrate control (30). Secretion was analyzed by immunoblotting using the introduced HA and FLAG epitopes at the C-termini of EsxA_1 and PPE68_1, respectively ( As several T7SS substrates, in particular those of the ESX-1 system, have been shown to be dependent on each other for secretion (30,32,33), we hypothesized that the secretion of heterologous EsxB_1/EsxA_1 might require the co-overexpression of the PE35/PPE68_1 pair that is putatively located in the same operon. In addition, similarly organized loci containing a pe/ppe pair and an adjacent esx gene pair can be observed in other ESX clusters. We co-electroporated the integrative pMV361::pe35/ppe68_1-flag and the pSMT3::esxB_1/esxA_1-ha in WT M. marinum. Secretion analysis followed by immunoblotting showed that the co-expression of EsxB_1/EsxA_1-HA did not seem to affect the expression and secretion of PPE68_1-FLAG ( Fig. 2A (Fig. 3).
These data show that the efficient secretion of overexpressed EsxA_1-HA relies on the cooverexpression of PE35/PPE68_1.
Because the integrative pMV361 plasmid and the multicopy pSMT3 plasmid differ in copy numbers, thereby possibly resulting in suboptimal co-secretion of the two substrate pairs, we also introduced the complete pe35/ppe68_1/esxB_1/esxA_1 locus into the pSMT3 plasmid again with a FLAG and HA tag fused to the C-termini of PPE68_1 and EsxA_1, respectively. We observed that while the cellular levels of both EsxA_1-HA and PPE68_1-FLAG was increased ( Fig substrates. This indicates that EsxB_1/EsxA_1 and PE35/PPE68_1 are co-dependently secreted via the ESX-1 system.  Interestingly, we observed that the presence of the SINGLE, DOUBLE and TRIPLE SWAP constructs seemed to cause some minor lysis of WT M. marinum cells, as a small amount of GroEL2 was consistently detected in the supernatants of these cultures (Fig. 4C). Nevertheless, the detected amount of GroEL2 was comparable among the strains expressing the different constructs, allowing further analysis. As observed before, the WT construct resulted in expression and secretion of both PPE68_1-FLAG and EsxA_1HA (Fig. 4C, lane 1-2 and 9-10). The EsxA antibody was included to confirm the total EsxA expression and secretion (Fig. 4C, lane 1-2 and 9-10). As seen previously for the SINGLE SWAP construct (30), we observed that PPE68_1 SWAP, appearing as a slightly higher PE/PPE determine system-specificity of Esx substrates 7 band than the PPE68_1 WT, was expressed and efficiently secreted in the WT strain (Fig. 4C, lane 3-4 and 11 -12). Notably, while secretion of PPE68_1 SWAP was more efficient than PPE68_1 WT (30), the amount of EsxA_1 was also higher in the supernatant fractions, as judged by an increased intensity of both HA and EsxA signals (Fig. 4C, lane [11][12]. The presence of the C-terminal tail of EsxM in the DOUBLE SWAP construct did not affect the secretion of both PPE68_1 SWAP and EsxA_1 as similar intensities of the detected signals were observed (Fig. 4C, lane [13][14]. However, when ESX-5 secretion signals were introduced in both PE35 and EsxB_1, the secretion of both the PPE68_1 SWAP and EsxA_1 seemed to reach the highest efficiency.

WT EsxB_1/EsxA_1 is rerouted to the ESX-5 system by introducing the EspG 5 binding domain in PPE68_1
We subsequently addressed the involved secretion systems by first introducing the different constructs in the ESX-1 mutant strain. In contrast to WT cells, GroEL2 was not detected in the supernatant fractions of this mutant strain, indicating the integrity of the cells in the presence of the constructs (Fig. 4D). As observed previously, secretion of both PPE68_1-FLAG and EsxA_1-HA of the WT construct was abrogated (Fig. 4D, lane 9-10). In contrast, the PPE68_1 SWAP protein was still secreted (Fig. 4D, lane [11][12], confirming our previous observation that the PPE68_1 SWAP was secreted independently from the ESX-1 system (30). Importantly, we also still detected EsxA_1 in the supernatant, using both the HA and the EsxA antibody (Fig. 4D, lane [11][12], suggesting that this ESX-1 substrate is secreted in an ESX-1 independent manner as well. This is highly interesting as both EsxA_1 and EsxB_1 are unmodified in the SINGLE SWAP construct. Similar as for the WT bacteria, the DOUBLE SWAP construct showed comparable levels of EsxA_1 secretion as the SINGLE SWAP constructs (Fig. 4D, lane 13-14), while the secretion of EsxA_1 seemed the most efficient in the presence of both the ESX-5 secretion signals in the TRIPLE SWAP construct (Fig. 4D, lane 15-16). Together, these data showed that PPE68_1 SWAP determines the ESX-1 independent secretion of EsxA_1.
To confirm that PPE68_1 SWAP and EsxA_1 are secreted by the ESX-5 system, we introduced the constructs in the ∆eccC 5 strain. EccC 5 is an essential component of the ESX-5 machinery (35,36) and deletion of this component blocks ESX-5-dependent secretion (7). In this strain, the presence of all tested constructs consistently caused minor bacterial lysis, as GroEL2 was found in all supernatant fractions (Fig. 4E). As a similar phenotype was observed for the SINGLE, DOUBLE and TRIPLE SWAP constructs in WT background, but not in the ESX-1 mutant strain, the bacterial leakage induced upon ectopic expression of these proteins seemed to be linked to a functional ESX-1 system. With the WT construct, we detected both PPE68_1-FLAG and EsxA_1-HA in the supernatant fractions by using the FLAG and HA antibody, respectively (Fig. 4E, lane 13-14).
The PPE68_1 SWAP and EsxA_1 of the SINGLE SWAP were moderately detected in the supernatant (Fig. 4E, lane 15-16), whereas they were no longer detected in the supernatants of bacteria containing either the DOUBLE or the TRIPLE SWAP (Fig. 4E, lane 17-20). In the two latter cases, the signals using the EsxA antibody were detected at comparable levels (Fig. 4E, lane 17-20) and were similar to that of the empty ∆eccC 5 strain (Fig. 4E, lane 3). Thus, our data show that the secretion of both PE/PPE determine system-specificity of Esx substrates 8 proteins became mostly dependent on the ESX-5 system when the EspG 5 binding domain was introduced. The observed residual secretion of PPE68_1-FLAG and EsxA_1-HA with the SINGLE SWAP construct indicates that a small amount of these substrate pairs is still secreted via ESX-1.
Interestingly, we previously showed that secretion of PPE68_1 SWAP was completely blocked in the same ESX-5 mutant in the absence of ectopically expressed EsxB_1/EsxA_1 (30). This indicates that this Esx substrate pair might be able to guide some amount of PPE68_1 SWAP to the ESX-1 system.
Finally, we observed a competitive correlation between the secretion of the rerouted substrates PE35/PPE68_1 SWAP and native substrates of the ESX-5 system, the PE_PGRS proteins.
Using the Genapol extraction method to analyse the surface localization of PE_PGRS proteins, we observed a lower amount of these proteins in the Genapol extracted fraction in the ESX-1 mutant strains expressing the four different constructs compared to WT bacteria containing the same constructs (Fig. 5). This suggests that, similar to what was reported previously, the redirection of ESX-1 substrates to the ESX-5 system interferes with the export of endogenous ESX-5 substrates (30).
In summary, introducing the EspG 5 binding domain in PPE68_1 resulted in the rerouting of both this PPE substrate and EsxA_1 to the ESX-5 system, not only further confirming that these proteins are co-secreted and but also showing that the PPE protein is involved in determining the system-specificity of the Esx substrate. Introduction of two ESX-5 secretion motifs optimized the secretion efficiency via ESX-5, showing that these signals have system-specific functionality to some extent. tuberculosis.

Discussion
In this study, we also confirmed that the C-terminal tails of PE35 and EsxB_1 are required for the secretion of the corresponding heterodimer, consistent with other studies (27). Moreover, our findings that these two secretion motifs are strictly required for the secretion of both heterodimers is highly interesting. These data show that the secretion of EsxA_1 is not only dependent on the cooverexpression, but also on the secretion of PE35/PPE68_1. In addition, the observation that secretion  (28,39,40). Specifically, the last seven amino acids of EsxB were shown to be essential for this interaction (28). The C-terminus of other Esx homologs in M. tuberculosis are likely to be structured similarly to the C-terminus of EsxB, but do contain different amino acids sequences, suggesting this domain might be involved in system-specific recognition (28,40,41). Importantly, structural analysis of EccC of Thermomonospora curvata showed that the crucial first NBD is kept in an inactivate state by a specific region in the linker 2 domain that connects the first and second NBD, and binding of EsxB is not able to activate this ATPase activity in vitro (40). It was therefore suggested that an additional trigger is necessary to activate EccC, which could be the binding of PE/PPE substrates. From our study, it seems evident that the secretion of the Esx substrates are closely linked to that of PE/PPE heterodimers. Possibly, they bind simultaneously or sequentially in order to activate all three ATPase domains of EccC, after which transport through the membrane complex is achieved. Such a model would explain both the necessity for equal expression levels of both heterodimers, as well as the secretion dependency of the Esx pair on the PE/PPE pair that we observed here. However, PE/PPE proteins can only be found in the genus of Mycobacterium, while the homologues of the Esx substrates and the EccC core component can be found in a more diverse repertoire of Gram-positive species (42)(43)(44). It will be interesting to see the differences in substrate recognition and secretion between these different systems.
The homologue of EsxA_1, EsxA, is the most-studied T7SS substrate and has been suggested to be responsible for ESX-1-induced phagosomal rupture. EsxA was found to be associated with membrane lysis when a transposon mutant of esxA/esxB was unable to lyse cultured lung epithelial cell lines (4,45). Further genetic studies in M. marinum showed that several different transposon mutants defective in EsxA secretion lost haemolytic activity and were attenuated in zebrafish (3,12,15,46), supporting the hypothesis that EsxA is a crucial virulent factor of pathogenic mycobacteria.
However, secretion of different ESX-1 substrate classes has been shown to be interdependent on each other (32,47), e.g. loss of EspA or PPE68 secretion led to secretion defects of EsxA and vice versa (30,32). Therefore, studying functions of individual ESX-1 substrates during the mycobacterial infection cycle has been a challenge. While protein sequences of EsxB and EsxB_1 are identical, EsxA_1 shares 92% protein sequence identity with EsxA. Given the high similarity, it has been suggested that EsxB_1/EsxA_1 have an equivalent functionality as the esx-1 encoded EsxB/EsxA (48). The observation that WT EsxA_1 can be destined for the ESX-5 system provides a unique platform to investigate exact roles of this protein in host-pathogen interactions. Current research is focusing on the redirection of EsxB/EsxA in order to directly assess the membrane lysis activity of this substrate pair.

Bacterial strains and growth cultures
All mycobacterial strains were grown on Middlebrook 7H10 plates (Difco) containing OADC supplement (oleic acid, albumin, dextrose and catalase; BD Biosciences) or liquid 7H9 medium containing ADC supplement (BD Biosciences) and the appropriate antibiotics (see below). M. marinum strains were grown at 30 °C, 90 rpm. All mycobacterial strains and mutants are listed in Table S1.
Escherichia coli strain DH5α was used for cloning procedures and plasmid accumulation, and was grown on lysogeny broth (LB) plates or liquid broth at 37 °C, 200 rpm. Growth media was supplemented with the appropriate antibiotics at the following concentrations: kanamycin (Roche) 25 µg/ml; hygromycin (Sigma) 50 µg/ml.

Plasmid construction
All PCRs were carried out with the Phusion High-Fidelity DNA polymerase (Finnzymes) using primers listed in supplemental Table S2. The restriction sites used for cloning are indicated in this table.     using Genapol X-080. Equivalent OD units were loaded; 0.2 OD for pellet and 0.5 OD for Genapol supernatants.