EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.


Introduction
Mycobacterium tuberculosis, the etiological agent for the disease tuberculosis (TB), is still one of the most dangerous pathogens for global health [1]. Successful infection requires secretion of multiple virulence factors, facilitated by type VII secretion systems (T7SS). Pathogenic mycobacteria have up to five T7SS, called ESX-1 to ESX-5 [2], of which at least three are essential for growth and/or virulence [3,4]. The ESX-1 locus was the first T7SS to be identified. The loss of ESX-1 function in Mycobacterium bovis BCG is considered a decisive factor of attenuation of this vaccine strain [5]. Mouse infection experiments utilizing M. tuberculosis with a partial deletion in ESX-1 showed reduced granuloma formation, the characteristic pathological hallmark of mycobacterial disease [6,7]. Similarly, efficient granuloma formation, dissemination of disease and invasion of endothelial cells in the fish-pathogen Mycobacterium marinum is dependent on a functional ESX-1 secretion system [8][9][10]. More detailed analysis showed that ESX-1 substrates are required for phagosomal membrane rupture [11,12].
Thus far, about a dozen different proteins have been identified to be secreted through ESX-1, which can be divided in three subgroups, the Esx proteins, the PE/PPE proteins and the Esp proteins. Of these substrates, the Esp proteins are ESX-1 specific [13]. The ESX-1 substrates EsxA (ESAT-6) and EsxB (CFP-10) are secreted as an antiparallel heterodimer [14]. Interestingly, the limited structural data available for PE and PPE proteins also show that these proteins form a heterodimer [15][16][17]. These heterodimers form a four-helix bundle and contain a YxxxD/E secretion motif directly after the helix-turn-helix on one of the Esx proteins and on the PE protein [15,18]. The ESX-1 substrate EspB forms a similar four helix bundle with the conserved secretion motif at the same position in the structure and therefore does not seem to require a partner protein [17,19]. EsxA and EsxB are most intensively investigated of the different ESX-1 substrates [11,[20][21][22] and EsxA is thought to be responsible for ESX-1 related virulence determinants [11,[21][22][23][24]. EspA and EspB have additionally been implicated to be important for virulence [25,26]. However, studying the exact role of each substrate is complicated, as deletion of esxA/esxB abolishes secretion of all different Esp proteins [8,27], while espA and espB deletion mutants are unable to secrete EsxA/EsxB [25,27].
The ESX-1 secretion system consists of a membrane complex composed of the ESX conserved components (Ecc) EccB 1 , EccCab 1 , EccD 1 and EccE 1 [28,29], which is stabilized by the MycP 1 protein [29]. The ESX-1 secretion system additionally contains the cytosolic accessory components EspG 1 and EccA 1 . EspG functions as a specific chaperone of cognate PE/PPE substrates [30,31] and deletion of espG 1 leads to a block in the secretion of PE35/PPE68_1 in M. marinum [31]. Loss of EspG 1 in M. tuberculosis caused severe attenuation, both in cell infection and in mice [32]. EccA 1 is a cytosolic AAA+ ATPase (ATPases Associated with diverse cellular Activities), which is essential for the EsxA secretion in both M. tuberculosis and M. marinum [33,34]. The M. marinum eccA 1 -null strain has been shown to be attenuated in zebrafish larvae [34]. However, its exact function is not further characterized.
In the M. marinum, the genes espG 1 (MMAR_5441) and eccA 1 (MMAR_5443) are separated in the esx-1 locus by espH (MMAR_5442). EspH-like proteins are unique for the ESX-1 system. EspD is a homologue of EspH, sharing 55% sequence identity in M. tuberculosis. EspD is encoded by the espACD locus, located more than 260 kb upstream of the ESX-1 gene cluster. Interestingly, M. tuberculosis EspD has a role in stabilizing the intracellular levels of the secreted substrate dimer EspA/EspC [35]. These observations suggest that EspH might function as a molecular chaperone.
Here, we study the role of three accessory proteins EspG 1 , EccA 1 and EspH in M. marinum and could show that mutants in the corresponding genes displayed distinctive and contrasting virulence phenotypes, demonstrating that ESX-1 substrates play different roles in virulence. We additionally identified several potential new ESX-1 substrates.

Individual ESX-1 components EspG 1 , EspH and EccA 1 , display distinctive effects on the secretion of ESX-1 dependent substrates
To study the role of accessory ESX-1 proteins EspG 1 , EccA 1 , and EspH in secretion, we created targeted knocked-out strains for espH and eccA 1 and used the previously described espG 1 knockout in M. marinum [31]. Deletion of the individual genes had no effect on bacterial growth in 7H9 medium (S1A Fig). However, colonies of the eccA 1 mutant appeared dry with a rough-surface, while no phenotypic change was observed for the ΔespG 1 and ΔespH colonies. In addition, qRT-PCR on total RNA extractions showed that the different deletions had no polar effect on the transcription of neighboring genes (S1B Fig). Next, secretion analysis was performed using immunoblotting and a set of antibodies directed against known ESX-1 substrates. GroEL2 was included as a loading and lysis control. As a known ESX-1 negative mutant we included the M vu strain, which has a frameshift mutation in eccCb 1 [4,36] (Fig 1B, lane 6 and lane 7, respectively). Our analysis showed that EsxA was no longer secreted in the ΔespG 1 strain (Fig 1B, lane 9), similarly as observed in a previous study from our group [31], but in contrast to the results obtained in M. tuberculosis [33]. Interestingly, the deletion of espH also resulted in a dramatic decrease in the secretion of EsxA ( Fig  1B, lane 10). Surprisingly, and in contrast to what has been published previously [8,34], we observed that secretion of EsxA was reduced in the eccA 1 mutant, but not completely aborted ( Fig 1B, lane 8).
Next, we analyzed another ESX-1 substrate EspE (MMAR_5439), a highly abundant cell surface protein of M. marinum, which can be extracted from the cell surface using the mild detergent Genapol X-080 [37]. The surface localization of the ESX-5 dependent PE_PGRS proteins was included as controls. In the WT strain, EspE was secreted in two forms: a full-length  protein of~40 kDa and a putatively processed form of~25 kDa (Fig 1C, lane 6). Surface localization of EspE was abolished in all the mutant strains ( Fig 1C, lane 7 to lane 10). Notably, while EspE accumulated in the cell pellet of all non-secreting strains, this protein was not detected in the pellet fraction of the espH mutant ( Fig 1C, lane 5), indicating that secretion of EspE was blocked at a different stage as compared to the other mutants.
To confirm that the observed secretion defects were caused by the targeted mutations, complementation plasmids were constructed. Two different complementation plasmids were used: the first one includes the genomic region from espF (MMAR_5440) to eccA 1 (MMAR_5443), whereas in the second plasmid only the espG 1 -espH-eccA 1 locus was present. Complementing the knockout strains with either of these plasmids fully restored the secretion of EsxA and EspE in all of the mutants (Fig 1D and 1E).

The absence of eccA 1 causes a loss of EsxA secretion under specific growth conditions
A major discrepancy with previous publications was our finding that EccA 1 has a limited effect on EsxA secretion. Previously, Gao et al. showed, using the same M. marinum background strain, that EccA 1 is crucial for ESX-1 secretion [8,34]. We realized that there is a difference in the growth conditions between the two studies; we used 7H9 medium whereas Gao et al. used Sauton medium [8,34]. To test whether the observed differences could be linked to a difference in growth condition, secretion analysis was performed on cultures grown in Sauton medium. Interestingly, whereas the results for ΔespG 1 and ΔespH were identical (Fig 2, lane 9 and lane 10, respectively), EsxA was no longer secreted in the eccA 1 mutant strain (Fig 2, lane 8), which shows that the role of EccA 1 in EsxA secretion is dependent on the growth condition.

Secretome analysis of accessory ESX-1 protein mutants by LC-MS/MS
The proteome of a number of ESX-1 targeted knockout strains of M. marinum has been determined previously [38]. However, this study did not include an espH mutant and the cell surface proteome was not analyzed. In order to obtain a comprehensive and detailed view, the complete secretomes of our mutant strains, the corresponding complemented strains and both the WT and ESX-1 secretion mutant eccCb 1 were analyzed by mass spectrometry. As some ESX-1 substrates are efficiently secreted into the culture supernatant, while others mainly remain attached to the cell surface [37], cells were grown with or without Tween 80 to study secreted proteins in the medium or the cell surface proteins, respectively. The cell surface proteins were extracted from the bacterial cells using Genapol X-080.
For the ESX-1 secretion (eccCb 1 ) mutant, a massive reduction in the secretion of all known ESX-1 substrates, i.e. EsxA (MMAR_5449), EsxB (MMAR_5450), EspB (MMAR_5457), EspC (MMAR_4167), EspE (MMAR_5439), EspF (MMAR_5440), EspJ (MMAR_5453), EspK (MMAR_5455) and PPE68 (MMAR_5448), was observed, both in the cell surface-enriched fractions ( Fig 3A) and the culture supernatants ( Fig 4A). These results are in line with published data [38]. Also the secretion of several other proteins, including the PE protein MMAR_2894 and PPE protein MMAR_5417, was blocked, suggesting they are novel ESX-1 substrates. This notion is strengthened by the fact that these two proteins are homologous to the PE and PPE protein encoded by the esx-1 locus. For the other proteins that showed marinum and the indicated mutants. In B we analyzed cell pellets not treated with detergent Genapol X-080 and culture supernatant fractions. In C we analyzed cell pellets treated with Genapol X-080 and the concomitant supernatant fractions. D and E. Complementation of the mutant strains fully restores ESX-1 secretion. In D the secretion of EsxA was analyzed and in E the secretion of EspE. In both experiments, GroEL2 was used as loading control and PE_PGRS as cell-surface control fraction. Equivalent OD units were loaded; 0.2 OD for pellet or Genapol pellet and 0.5 OD for supernatant or Genapol supernatant fractions. reduced spectral counts in the cell surface fractions it is more difficult to draw any conclusion. First of all, the difference in secretion levels are smaller as compared to the known ESX-1 substrates (Fig 3), but furthermore they lack known characteristics of T7SS substrates, such as the YxxxD/E secretion motif preceded by a predicted helix-turn-helix structure. The espG 1 mutant showed similar secretion profiles as the eccCb 1 mutant (Fig 3B and Fig 4B), although the secretion of EspB, EspK and EspE seemed to be slightly less severely affected. This suggests that EspG 1 is not only required as a chaperone for its cognate PE/PPE substrates, but plays a more central role in the secretion of all ESX-1 substrates. The secretion of all ESX-1 substrates returned to WT levels in the espG 1 mutant carrying the pMV361::espF-eccA 1 complementation plasmid (S2A and S2B Fig).
The secretome profiles of the eccA 1 mutant in 7H9 medium showed only a mild reduction of ESX-1 substrates in both cell surface and supernatant fractions (Fig 3D and Fig 4D). For instance, EsxA and EsxB secretion was five and two-fold decreased, respectively, while in the eccCb 1 mutant the reduction of both was 10 fold (Fig 4D). The substrates EspE, EspF, EspJ and EspK are more affected by the eccA 1 mutation than the other substrates in both protein fractions. In concordance with the data obtained by immunoblotting, the complementation of the eccA 1 mutant with pMV361::espF-eccA 1 plasmid restored the secretion of all ESX-1 substrates (S2A and S2B Fig).
Deletion of espH resulted in a severe reduction of EspE and EspF (Fig 3C), in line with our immunoblot analysis. This reduction was in fact almost complete, both in the fraction of the This effect was restored when the complementation plasmid was introduced (S2A and S2B  Fig). Interestingly, the effects of the espH deletion on secretion of EsxA and EsxB was only mild as compared to the eccCb 1 mutant, while the effects on other ESX-1 substrates, such as EspB, EspK and EspJ were also only minor (Fig 4C). This indicates that ΔespH has a specific Differential roles of ESX-1 substrates in mycobacterial infection secretion defect for a subset of ESX-1 substrates and there is no substrate dependency between EspE/EspF and other Esp proteins.
Surprisingly, we also identified some proteins that were present in significantly increased amounts in the cell surface enriched fractions of various mutants. One of these proteins is SecA2, a cytosolic component of the Sec transport system and proposed to contribute to the virulence of M. tuberculosis and M. marinum [39,40]. SecA2 was present in higher amounts in all mutants except the ΔespH, suggesting a link with intracellular accumulation of EspE/EspF. Another intriguing observation is an increase of Mak in the ΔespG 1 , ΔespH and the ΔeccA 1 ( Fig  3B, 3C and 3D, respectively). Mak is a mycobacterial maltokinase whose function is involved in the glycan synthesis from trehalose [41] and considered to be essential for the growth of M. tuberculosis [42]. This could suggest that there is an indirect effect of ESX-1 secretion on the synthesis of the mycobacterial capsule.

EspE specifically interacts with EspH in M. marinum
The observation that EspH mainly affects the secretion of EspE/EspF and that EspE could not be detected in the espH mutant pellet fraction raised the hypothesis that EspH could either regulate the transcription of espE/espF or stabilize EspE/EspF at the protein level. To get more information on the putative function of EspH we used the protein structure prediction program Phyre2 [43]. This analysis showed that part of EspH (region between amino acid 65 and 135) is predicted to share structural similarity to YbaB proteins of Escherichia coli and Haemophilus influenza. Although the sequence identity with these proteins is low (15%) the confidence of the structural homology is very high (97%). Because YbaB is reported to be a small DNA-binding protein that plays a regulatory role [44], an effect on transcription regulation could be possible. Therefore, we measured the effect of espH deletion on espE and espF mRNA levels. Because the EsxA secretion was reduced in the espH mutant, esxA mRNA level was checked as well. Total mRNA was extracted from the WT M USA , eccCb 1 mutant and the ΔespH strain, and qRT-PCR was performed using primer sets for espE, espF and esxA. The results showed that the mRNA levels of all three genes were comparable to those of the eccCb 1 mutant strain analyzed (S3A Fig). Thus, we could disprove the possibility that EspH regulates espE at the transcriptional level.
Next, we studied a direct interaction of EspH with EspE and/or EspF. Based on the high homology of EspE with EspA and EspF with EspC, we speculated that, similarly to EspC/EspA [45], EspF might be secreted together with EspE. We therefore constructed a plasmid containing espE/espF in which espE was modified to express a C-terminal Strep tag. We also introduced a His tag at the C terminus of EspH in the espG 1 /espH/eccA 1 complementation plasmid. Introduction of both plasmids in the WT and ΔespH mutant resulted in surface localized EspE, as judged by immunoblot analysis of the cell surface extracted protein preparations (S3B Fig). These results show that the addition of the Strep tag to the C terminus of EspE and the His-tag to EspH did not affect the functionality of these proteins in the secretion process.
To study the interaction of EspE and EspH, we overexpressed EspE-Strep/EspF and EspH-His in the eccCb 1 mutant strain. The ESX-1 secretion system is defective in this strain and therefore EspE and EspH accumulate in the cytosol, which allows their analysis and co-purification. The subcellular localization of EspE and EspH was examined by a subcellular fractionation procedure, showing that EspE-Strep was partially soluble while EspH-His was exclusively present in the soluble fraction (S3C Fig). Next, we used StrepTactin beads to purify Strep-tagged EspE from these soluble fractions. Immunoblot analysis showed that EspE-Strep was efficiently purified. Importantly, EspH-His, appearing as a~25 kDa band, was only present in the elution fractions when expressed in the presence of EspE-Strep ( Fig 5A). In contrast, the ESX-1 substrates PPE68 and EsxA were not co-purified and both remained in the flowthrough fraction.
To confirm this EspE-EspH interaction, a reciprocal pull-down assay was performed using Ni-NTA beads and lysates of the eccCb 1 mutant containing EspE-strep/EspF only or EspEstrep/EspF and EspH-His. Immunoblot analysis confirmed the efficient purification of EspH--His ( Fig 5B). Using anti-EspE on these samples showed co-elution of endogenous EspE only in the presence of the His-tagged EspH ( Fig 5B). Again, PPE68 and EsxA were only found in the flow-through fraction, indicating that they do not bind EspH. In conclusion, these data confirmed that EspH specifically interacts with EspE in the cytosol of M. marinum and this interaction is probably required for EspE secretion.

The espH mutant is attenuated in phagocytic cells and shows strongly reduced hemolysis
ESX-1 functioning in M. marinum has been associated with lysis of red blood cells [8]. Because of this, the hemolysis assay has been employed as a model for the ESX-1-dependent lysis of (phagosomal) membranes [8]. Prior work suggested that the ESX-1 associated membrane lytic activity was mediated by EsxA through its pore-forming activity [21,46]. Because the deletion of espG 1 , espH and eccA 1 differently affected the secretion of EsxA, we examined to what extend these mutant strains were able to disrupt erythrocytes. While we confirmed that our WT strain showed hemolysis (Fig 6A), both the eccCb 1 and ΔespG 1 mutant strain lost this ability, in line with the absence of ESX-1 substrates in the culture supernatant ( Fig 6A). Interestingly, the ΔespH and ΔeccA 1 strains were also non-hemolytic, although these strains were still able to secrete EsxA to significant levels ( Fig 6A). The defects in hemolysis by the knockout strains were restored when the complemented plasmids were introduced into these mutant strains ( Fig 6B). As in the ΔespH and ΔeccA 1 mutants mainly the secretion of different Esp proteins are specifically affected, our findings indicate that not a single ESX-1 substrate, such as EsxA, but a combination of different Esp proteins, are responsible for the hemolytic phenotype.
To further characterize the function of the different ESX-1 substrate subsets, we used different phagocytic cells to study the ability of the mutant strains to survive and replicate within these cells. Phagocytic cells from mice (RAW macrophage cell line) and the protozoa Acanthamoeba castellanii were infected with green fluorescent protein (GFP)-expressing mycobacteria and infection levels were quantified by flow cytometry at different time points. As shown before, the eccCb 1 mutant was strongly attenuated in both A. castellanii and RAW cells (Fig 7; [47]), showing a 2-fold reduction in the number of infected cells after 24 h. As expected, based on the proteome profiles, the ΔespG 1 strain showed an attenuated phenotype similar to the eccCb 1 mutant. For the ΔespH mutant, the proportion of infected A. castellanii cells did not change over time (Fig 7B), while in RAW macrophages a slight reduction of infected cells at 24 hpi could be observed (Fig 7C, p = ns). Infection with the ΔeccA 1 mutant resulted in an increase of infected cells over time, for both A. castellanii and RAW cells, and was therefore less attenuated as compared to the other mutants (Fig 7B and 7D). Although this strain was able to infect A. castellanii to the same extend as the WT strain, infection with this mutant was not as successful as WT infection in RAW macrophages (Fig 7A, ns; Fig 7C, p < 0.001).
Taken together, our data show the importance of espG 1 in achieving successful infection of phagocytic cells, while the loss of eccA 1 only marginally affects the ability of M. marinum to survive and replicate in a phagocytic host cell. These findings are in line with the proteomic analysis, i.e. the espG 1 mutation has a strong effect on secretion of all ESX-1 substrates, while deleting eccA 1 only results in a mild secretion defect. EspH, which seems to mainly influence EspE and EspF secretion, is also important for infecting phagocytes, but to a lesser extent than EspG 1 . Together, this shows that espG 1 , but not eccA 1 , plays a major role in early stages of infection in vivo. Moreover, since these strains show a comparable behavior during in vitro and in vivo infections, this indicates functional similarities for these genes in protozoa, mouse macrophages and zebrafish larvae.

The absence of espH results in a hypervirulent phenotype in zebrafish larvae
In contrast to the ΔespG 1 and ΔeccA 1 strain, the behavior of ΔespH in zebrafish larvae was completely different from its attenuated phenotype in vitro. Systemic infection of zebrafish larvae resulted in an increased bacterial load as compared to WT infection (Fig 8C; p < 0.05). Large bacterial clusters and a phenotype known as cording were seen in fluorescence images (Fig 8L, arrow), especially at higher magnification of individual clusters (Fig 8L, closed arrow, n = 15 larvae). Cording in zebrafish has been associated with extracellular growth [48]. In addition, very limited numbers of intact phagocytic cells and the presence of fluorescent spots suggestive for phagocytic cell debris were observed (Fig 8L, open arrow).
These observations raised the question whether this phenotype is still preceded by granuloma formation or if this mutant strain is preventing early granuloma formation by inducing rapidly host cell death. Therefore, larvae were systemically infected with either ΔespH or WT M. marinum as control and monitored daily for 4 consecutive days (Fig 9). Mycobacteria were phagocytosed by L-plastin positive phagocytic cells at 1 dpi in both groups (Fig 9A and 9D). Subsequently, phagocytic cells were recruited and early granulomas started to form (Fig 9B  and 9E). However, at 4 dpi, in larvae infected with the ΔespH strain a strong decrease in phagocytic cells and increase in bacterial growth was observed (Fig 9C and 9F). In the absence of phagocytic cells bacteria were able to show cording in both blood vessels (Fig 9F, closed arrow) and tissue (Fig 9F, open arrow). Taken together, the ΔespH mutant seems to have a host-specific or in vivo-specific effect, illustrated by a hypervirulent phenotype seen in zebrafish larvae, but not in cell infections in vitro. Therefore, our data indicates that EspH is not required for initial phagocytosis, recruitment of cells and primary establishment of early granulomas, but this protein, and therefore a subset of ESX-1 substrates, seems to be essential for the maintenance of a stable granuloma.

Discussion
A number of studies have shown that the mycobacterial ESX-1 system plays a pivotal role in mycobacterial pathogenesis [6,21,27,33]. The system affects virulence through secretion of protein effectors with host-modulatory effects. Here, we show that EccA 1 is not strictly required for the secretion of ESX-1 substrates. The finding that EccA 1 is important for secretion is in line with previous reports [8,34], but the fact that the role of EccA 1 is depending on the growth medium is entirely surprising. This difference could also explain the variable results described for the role of EccA 1 in EsxA secretion by M. tuberculosis [49]. Of all ESX-1 substrates, EspE, EspF, EspJ and EspK secretion was mostly affected in our eccA 1 mutant strain, while secretion of EspB, EsxA/EsxB and PE/PPE was hardly altered. An interesting observation here is the discrepancy between the active secretion of EsxA in the ΔeccA 1 strain and at the same time loss of hemolytic activity. Although this observation has been described before, this was always linked to a reduced secretion of EsxA in these strains [8,34]. In a recent study, the importance of EsxA in lysing membranes was questioned [50]. Our results also supports an alternative mechanism: we find a strong correlation between ESX-1 functionality and Differential roles of ESX-1 substrates in mycobacterial infection hemolysis, but this correlation is not seen for EsxA secretion. Our finding is in line with several other recent studies, who showed that both EsxA and the cell-surface lipid PDIM are important for phagosomal rupture and escape by M. tuberculosis [51][52][53]. We propose that it is not the loss of secreted EsxA, but the loss of (multiple) surface-exposed Esp proteins that results in hemolytic deficiency.
Even though the ΔeccA 1 strain lost its ability to induce membrane lysis, virulence in isolated phagocytes and in zebrafish larvae was only mildly affected in our study. This is in contrast with other studies, who described an attenuated phenotype for similar mutants in M. tuberculosis and M. marinum in murine macrophages and zebrafish [8,34]. The latter observations were made after a longer incubation time, which might explain the discrepancy with our study. Distinct phenotypes of the eccA 1 mutant in different host cells have also been reported in a genome-wide transposon mutagenesis study [47]. Here, transposon insertions in M. marinum E11 eccA 1 led to severe attenuation in mammalian phagocytic cells but these mutants were hypervirulent in protozoan cells [47]. This suggests that M. marinum can employ host-specific virulence mechanisms to adapt to different intracellular environments and that EccA 1 might be essential for secretion and virulence under specific circumstances or in a subset of specific hosts.
The role of EspG as a specific chaperone for the recognition and secretion of cognate PE/ PPE proteins has been well established in M. marinum [30,31]. Our extracellular proteomic study not only confirms the loss of PE/PPE substrate secretion in the M. marinum ΔespG 1 strain, but also reveals the secretion block of other ESX-1 dependent substrates, including EsxA/EsxB. This effect on EsxA/EsxB secretion however was not observed in an M. tuberculosis espG 1 knock-out strain [33]. EspG proteins bind specifically to the extended helices of the PPE protein, which are absent in Esx proteins. Therefore, the strong effect of espG 1 deletion on Esx (and also Esp) protein secretion in M. marinum is likely indirect due to a mutual dependency in secretion among the ESX-1 substrates [27,31,35]. This co-dependency of PE/PPE and other ESX-1 substrates for secretion is possibly less strict in M. tuberculosis, explaining that mutating espG 1 had no effect on EsxA/EsxB secretion in this species. Because of the severe secretion defect of all detectable ESX-1 substrates, the M. marinum espG 1 mutant is non-hemolytic and strongly attenuated in macrophage and amoeba, which is in good agreement with previous reports [8]. Furthermore, the loss of espG 1 resulted in a strong attenuation in zebrafish, to the same extend as the eccCb 1 mutant.
Our most significant and surprising results were obtained for EspH. EspH is specific for the ESX-1 secretion system and is highly conserved among pathogenic mycobacterial species, including M. tuberculosis and M. leprae. The latter species has been streamlined into a minimal genome by a process of extensive genome decay. In our study, deletion of this gene abolishes the expression and secretion of two specific ESX-1 substrates EspE and EspF. Furthermore, we could show that EspH specifically interacts with EspE in the cytosol, indicative of chaperone activity. However, the Phyre2 structural prediction program [43] indicated that EspH is shares similarity to YbaB. The first structural study on YbaB strongly indicated that this protein binds DNA as a dimer [44]. However, recent studies indicated that the function of YbaB might be more diverse. One study showed that YbaB is associated with and a target of ClpYQ proteases in E. coli [54], while another study indicated that overexpression of YbaB enhanced the production of heterologous membrane proteins [55]. Based on the direct interaction of EspH with EspE and that the EspH-like protein EspD stabilizes intracellular EspA/EspC substrates [35], we propose that these YbaB-like proteins encoded by the esx-1 cluster of pathogenic mycobacteria function as dedicated chaperones for specific ESX-1 substrates. Recently, a study of M. tuberculosis EspL also predicted a high resemblance to YbaB [56], making it tempting to speculate that EspL may as well function as a dedicated chaperone, for instance the ESX-1 substrates encoded by the adjacent genes EspK or EspB. It becomes clear that multiple chaperones, such as EspG 1 , EspD and EspH, are responsible for stabilizing their cognate substrates PE35/PPE68, EspC/EspA and EspF/EspE, respectively. Interestingly, secretion of other substrates of the ESX-1 system, such as EspB, EspK and EspJ, did not seem to be affected by disruption of the espH gene. A similar phenotype was observed previously in an espA::tn mutant of M. tuberculosis [26], where secretion of EsxA/EsxB but not EspB was aborted. These results show that interdependence in ESX-1 secretion is not a general feature. Deletion of espH did result in reduced secretion of EsxA/EsxB, which was not due to differences in mRNA levels. This hints towards a possible regulation mechanism between the secretion of the central components EsxA/EsxB and the individual Esp substrate (pairs) but not among the Esp proteins themselves.
The espH mutant strain showed a loss of hemolytic activity and a reduction of intracellular growth in phagocytic host cells in our study. Strikingly, zebrafish larvae were heavily infected with this mutant strain and showed even hypervirulence at later time points, even though EsxA/EsxB secretion was reduced in this mutant. More detailed analysis showed that initial phagocytosis and primary establishment of an early granuloma was not affected in this mutant. Eventually, a stable cluster of immune cells could not be maintained in larvae infected with the espH mutant, with subsequent extracellular bacterial outgrowth and apparent phagocyte death. The discrepancy between in vitro and in vivo results indicate an essential role for a, yet unknown, host factor involved. It is tempting to speculate that EspE/EspF, the two proteins that are most severely affected by the espH deletion, interact with this host factor in order to induce the homeostatic balance between host and pathogen in developing granulomas. Furthermore, because EsxA and EsxB secretion was diminished, other ESX-1 substrates in addition to these central substrates might be involved in the infection process. A candidate might be EspB, whose secretion was not affected in espH mutant strain, and was shown to be able to facilitate M. tuberculosis virulence in vitro and in vivo in an EsxA-independent way [26].
In summary, this study highlights the complexity of the ESX-1 secretion machinery. We unravel valuable information about the functions of the individual ESX-1 components EccA 1 , EspG 1 and EspH, all having their unique role in secretion of the different substrate classes. We can conclude that ESX-1 has several different sets of substrates that are involved in distinctive processes required for virulence.

Bacterial strains and cell cultures
All M. marinum strains used in this study were derived from the wild-type strain M USA [57]. The eccCb 1 (M VU ) strain was previously identified as an ESX-1 secretion mutant with a spontaneous out of frame mutation in eccCb 1 [36] and also the knock-out strain espG 1 was described before [31]. The knockout strains of eccA 1 and espH were generated using the mycobacteriophage approach (see below). All strains were routinely cultured on Middlebrook 7H10 plates or in Middlebrook 7H9 medium (Difco) containing ADC supplement or on Sauton medium [58] supplemented with 2% glycerol and 0.015% Tween-80. When required, 0.05% Tween-80 and the appropriate antibiotics were added (25 μg/ml kanamycin (Sigma) and/ or 50 μg/ml hygromycin (Roche). M. marinum cultures and plates were incubated at 30˚C. E. coli TOP10F' was used for cloning experiments to generate the complemented plasmids and was grown at 37˚C on LB plates and in LB medium. Different antibiotics were added to the cultures or plates when necessary at similar concentrations as for M. marinum cultures.

DNA manipulation and plasmid construction
All DNA manipulation procedures followed standard molecular biology protocols. Primers were synthesized and purified by Sigma. Phusion polymerase, restriction enzymes and T4 DNA ligase were obtained from New England Biolabs (NEB). Macrogen performed DNA sequencing.

RNA extraction and RT-PCR analysis
Bacterial RNA was extracted from various M. marinum strains as described previously [31] and cDNA was synthesized using SuperScript VILO cDNA Synthesis kit (Thermoscientific) according to manufacturer protocol. For the PCR mix the SYBR GreenER qPCR SuperMix (Thermoscientific) was used according to manufacturer instructions, including the addition of ROX dye reference. qRT-PCR was performed in Applied Biosystems 7500 Fast system. The primer sequences used for qRT-PCR are listed in S3 Table. Controls without reverse transcriptase were done on each RNA sample to rule out DNA contamination. The sigA gene was used as an internal control.

Generation of the knockout strains
An eccA 1 and espH knockout was produced in M. marinum M USA by allelic exchange using a specialized transducing mycobacteriophage as previously described [59]. High phage titers were prepared following the previously described protocol [31]. Subsequently, the M. marinum wild-type strain was incubated with the corresponding phage to create eccA 1 and espH knockouts. Colonies that had deleted the endogenous eccA 1 and espH were selected on hygromycin plates and tested for sucrose sensitivity, induced by the presence of the sacB gene. The deletion was confirmed by PCR analysis and sequencing. To remove the resistance and sacB gene, a temperature sensitive phage encoding the γδ-resolvase (TnpR) (a kind gift from Apoorva Bhatt, University of Birmingham, UK) was used, generating an unmarked deletion mutation.

M. marinum secretion analysis and immunoblotting
M. marinum cultures were grown in 7H9 medium supplemented with ADC and 0.05% Tween 80 to mid-logarithmic phase. Bacteria were washed two times and set to OD600 of 0.35 in 7H9 medium containing 0.2% glycerol, 0.2% dextrose and 0.05% Tween 80 for overnight growth. Supernatants were filtered using 0.2 μm filter, concentrated by trichloric acid (TCA) precipitation, washed with acetone and the supernatant pellets were resuspended in solubilisation/ denaturation (S/D) buffer (containing 100mM DTT and 2% SDS). Bacteria were washed once with PBS. Aliquots were taken for the whole cell lysate preparations and for Genapol X-080 extraction of cell-surface-attached proteins. Bacteria were incubated with 0.5% Genapol X-080 in PBS for 30 minutes with head-over-head rotation at room temperature. Genapol extracted supernatants were denatured in S/D buffer. The bacterial pellet and Genapol extracted cells were lysed by bead beating for 1 minute two times after which S/D buffer was added. All samples were boiled for 10 minutes at 95˚C before loading on SDS-PAGE.

Pulldown assays
For His-tag pulldown, mycobacterial cultures grown to an OD600 of 1.0 were incubated for 1 h with 100 g/ml ciprofloxacin (Sigma), pelleted, washed twice with PBS, and subsequently resuspended in PBS supplemented with Complete protease inhibitor mixture (Roche Applied Science), 1 mM EDTA, and 10 mM imidazole. Cells were broken by two-times passage through a One-Shot cell disrupter (Constant Systems) at 0.83 kbar. Unbroken cells were spun down by repeated centrifugation at 3000 g, and subsequently the cell envelope and soluble fractions were separated by ultracentrifugation at 100,000 g for 1hr. Membrane-cleared lysates of M. marinum expressing proteins of interest were incubated with Ni-NTA agarose beads (Qiagen) for 1 h at room temperature with head-over-head rotation. After washing the beads five times with phosphate buffer containing 50 mM NaH 2 PO 4 and 300 mM NaCl, (pH 8.0), supplemented with 20 mM imidazole, bound proteins were eluted three times by incubation with phosphate buffer containing 400 mM imidazole. Immunoprecipitation of strep-tagged proteins was performed using the Strep-Tactin Sepharose kit (IBA), following the manufacturers protocol.

LC-MS/MS
To investigate the cell-surface attached proteome, samples for LC-MS/MS analysis were prepared using the mild detergent Genapol X-080 as previously described [63]. To prepare the secreted material, the M. marinum M USA wild-type and the studied ESX-1 mutant and complemented strains were grown to stationary phase in 7H9 medium supplemented with ADC, 0.2% glycerol and 0.05% Tween 80. The supernatant fractions containing secreted proteins were collected and spun at 2500 × g for an additional 20 min at 4˚C and subsequently filtered through a 0.2 μm pore size membrane to remove residual cells and cell debris. The filtered supernatants were 20 times concentrated using Amicon Ultra-15 Centrifugal 3 kDa molecular weight cut off membrane at 4˚C. The retained proteins were TCA precipitated, pelleted, washed in acetone, dried and resuspended in S/D sample buffer to the corresponding OD of 200 units/ml. All samples were analyzed with SDS-PAGE and CBB staining. Total protein lanes of cell surface and culture supernatant proteins were excised in 3 or 1 fragment(s) per lane, respectively, and analyzed by LC-MS/MS as described before [63]. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008905.

Hemolysis assay
M. marinum strains were grown in 7H9 medium supplemented with ADC and 0.05% Tween-80 till the mid-logarithmic phase. All strains were washed once with PBS and inoculated in 7H9 medium with or without Tween-80 at 0.35 OD600/ml and inoculated for 20 hours. Bacteria were collected by centrifugation, washed once in PBS and diluted in fresh DMEM medium without phenol red (Gibco, Life technologies). Bacteria were quantified by absorbance measurement at OD600 with an estimation of 2.5 Ã 10 8 bacteria in 1 ml of 1.0 OD 600 . At the same time, defibrinated sheep erythrocytes (Oxoid-Thermo Fisher, the Netherlands) were washed five times and diluted in the same fresh DMEM medium. 4.2 Ã 10 7 erythrocytes and 1 Ã 10 8 bacteria were added for one reaction of 100 μl in a round-bottom 96 well-plate, gently centrifuged for 5 minutes and incubated at 32˚C. After an incubation of 3 hours, cells were resuspended, centrifuged and 80 μl of supernatants were transferred to a flat-bottom 96-wells plate and measured at an absorbance of 405nm to quantify hemoglobin release.

Host cell infection procedure
All bacterial strains were grown until the exponential growth phase, washed with 0.05% Tween 80, spun down and resuspended in RPMI medium. RAW macrophages were infected with a MOI of 5 for 3 hours and incubated at 30˚C, 5% CO 2. Cells were washed in RPMI to remove extracellular mycobacteria and either analyzed immediately or incubated for another 21 hours at 30˚C, 5% CO 2. A. castellanii (ATCC 30234) infection was performed with a MOI of 1, 3, 9, 27, 54, and 108 to determine optimum MOI, for the remaining experiments MOI 3 was chosen. Incubation for 3 hours or 24 hours was done at 30˚C, 5% CO 2.

Flow cytometry
Uptake of strains in host cells was quantified for all cell lines with a BD Accuri C6 flow cytometer (BD Biosciences) with a 488-nm laser and 585/40-nm filter to detect mEos3.1. A minimum of 5000-gated events was collected per sample per time point, percentage of living cells, percentage of infected cells and median fluorescent intensity per cell was analyzed using BD CFlow software.

Injection stocks for zebrafish infection
Injection stocks were prepared by growing bacteria until the logarithmic phase (OD 600 of 0.7-1). Bacteria were spun down at low speed for 1 min to remove the largest clumps, washed with 0.3% Tween-80 in phosphate buffered saline (PBS) and sonicated shortly for declumping. Bacteria were than resuspended in PBS with 20% glycerol and 2% PVP and stored at −80˚C. Before use, bacteria were resuspended in PBS containing 0.17% (V/V) phenol red (Sigma) to aid visualization of the injection process.

Zebrafish infection procedure
Transparent casper zebrafish larvae [64] were removed from their chorion with tweezers and infected at 1 day post fertilization (dpf) via the caudal vein with bacterial suspension containing 50-200 CFU. Injection was performed as described previously [65]. To determine the exact number of bacteria injected, the injection volume was plated on 7H10 plates containing the proper antibiotic selection. At 4 days post infection (dpi) larvae were analyzed with a Leica MZ16FA fluorescence microscope. Bright field and fluorescence images were generated with a Leica DFC420C camera. Infection levels were quantified with a custom-made fluorescent pixel counting software. The software is in house developed and freely available under MIT license. Following analysis, larvae were fixed overnight in 0.4% (V/V) paraformaldehyde (EMS, 100122) in PBS, washed and stored in PBS for immunohistochemistry.

Ethics statement
All procedures involving Danio rerio (zebrafish) larvae were performed in compliance with local animal welfare laws and maintained according to standard protocols (zfin.org). The breeding of adult fish was approved by the institutional animal welfare committee (Animal Experimental licensing Committee, DEC) of the VU University medical center. All protocols adhered to the international guidelines specified by the EU Animal Protection Directive 86/ 609/EEC, which allows zebrafish larvae to be used up to the moment of free-living (approximately 5-7 days after fertilization). In the current study, zebrafish larvae were used between 1 and 5 days post fertilization.

Confocal microscopy
Confocal analysis was performed on larvae, embedded in 1% low melting-point agarose (Boehringer Mannheim, 12841221-01) in an 8-well microscopy μ-slide (ibidi), Analysis was performed with a confocal laser scanning microscope (Leica TCS SP8 X Confocal Microscope). Leica Application Suite X software and ImageJ software were used to adjust brightness and contrast and to create overlay images and 3D models.

Graphs and statistical analysis
Graphs were made using Graph Pad Prism 6.0. Pixel counts were logarithmic transformed; error bars represent mean and standard error of the mean. A one-way ANOVA was performed followed by a Bonferroni's multiple comparison test to analyze statistical significance. Graphs with results of RAW264.7 and A. castellanii infection experiments show percentage-infected cells of total cells; error bars represent mean and standard error of the mean. Data representing the fold change between 3 and 24 hpi was logarithmic transformed. A two-way ANOVA followed by a Sidak's multiple comparison test was performed for statistical significance.