A Mycobacterium ESX-1–Secreted Virulence Factor with Unique Requirements for Export

Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.


Introduction
The cell surface-associated and secreted proteins of pathogenic bacteria promote the uptake of nutrients; facilitate attachment to specific surfaces, cells, or proteins; function in cell wall maintenance and cell division; and offer protection from harsh environmental conditions, including the host immune system. In Mycobacteria, there are at least four pathways to secrete proteins-Sec, SecA2, twin-arginine translocase, and the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1). Much attention has been focused on the ESX-1 pathway because it is required for virulence and for the secretion of ESAT-6 and culture filtrate protein 10 (CFP-10), two major targets of the immune response in infected individuals.
M. tuberculosis ESX-1 is required for virulence in mice, growth in macrophages, and the suppression of macrophage inflammatory and immune responses, including the arrest of phagosome maturation and the reduced expression of IL-12 and TNF-a [1][2][3][4][5][6]. The homologous M. marinum ESX-1 is required for virulence in zebrafish, growth in macrophages, cytolysis and cytoxicity, and cell-to-cell spread, in addition to ESAT-6 and CFP-10 secretion [7,8]. In zebrafish embryo infections, M. marinum ESX-1 is required for macrophage aggregation and granuloma formation [9]. In M. smegmatis, ESX-1, in addition to being required for secretion of ESAT-6 and CFP-10, modulates conjugal DNA transfer [10,11]. In contrast, most strains of M. ulcerans, which is closely related genetically to M. marinum and M. tuberculosis, but persists in extracellular locations during mammalian infection, lack most of the ESX-1 components as well as orthologs of the genes extending from Rv3879c thru Rv3883c [12,13]. Although the ESX-1 secretion machinery (Rv3870, Rv3871, and Rv3877) is required for the arrest of phagosome maturation by M. tuberculosis during an infection of macrophages, the known ESX-1 substrates are dispensable [6]. The multiple phenotypes and host responses dictated by the ESX-1 secretory apparatus suggest that there may be additional substrates, components, and regulatory molecules yet to be identified.
Recently, a third ESX-1 substrate, EspA (Rv3616c), was identified [14]. Unlike ESAT-6 and CFP-10, EspA is encoded at a locus distant from the ESX-1 machine, yet this substrate is codependent with both ESAT-6 and CFP-10 for secretion. The mechanism for this interdependence has not been determined, but the interaction between ESAT-6 and CFP-10 in the bacterial cytosol appears to be required for secretion of the heterodimer [15][16][17][18]. Presumably, the stable heterodimer is also required for the secretion of EspA.
The M. tuberculosis region of difference 1 (RD1) locus (Rv3871-Rv3879c) and the neighboring genes encode the ESX-1 substrates ESAT-6 and CFP-10, as well as core components of the secretion machine [1][2][3]. These core components include at least two putative SpoIIIE/FtsK ATPase family members (Rv3870 and Rv3871), a proline-rich predicted chromosome-partitioning ATPase (Rv3876), and a putative transporter protein with 12 transmembrane domains (Rv3877). The non-RD1 gene cluster Rv3616c-Rv3614c also is required for secretion of the known substrates [14,19]. Additional proteins are likely to be necessary for the assembly of the ESX-1 machinery, because in M. smegmatis, genes extending from homologs of Rv3866 through Rv3883c have been shown to be required for ESX-1-mediated secretion [11]; an M. bovis mutant disrupted for the expression of the genes homologous to Rv3867 through Rv3869 fails to secrete ESAT-6 and CFP-10 [20]; and in M. marinum, the locus required for ESX-1-mediated secretion extends at least from the homolog of Rv3866 (Mh3866) to the homolog of Rv3881c (Mh3881c), which in this work we rename espB (see below) [7].
Although these studies have identified multiple genes required for ESX-1 function, the biochemical interactions necessary for assembly of the secretion machine and for transport of substrates are still not understood. A model for CFP-10 secretion is that the carboxyterminus of the CFP-10 substrate is recognized by Rv3871, which in turn interacts with the integral membrane protein Rv3870 to direct CFP-10 through the secretion pore [15]. The interaction of CFP-10 with Rv3871 is also required for secretion of ESAT-6, suggesting that this is a requisite step in secretion of the ESAT-6/CFP-10 heterodimer by the ESX-1 machine.
Here, we show that Rv3881c and its M. marinum homolog, Mh3881c, are substrates for secretion by ESX-1. For this reason, we have named the gene product of this locus ESX-1 substrate protein B (EspB). In both species, espB encodes a gylcine-rich protein with a predicted molecular weight of ;47 kDa, without any region of apparent similarity to the secretion signal of CFP-10 or other known secretion signals. Although a substrate of ESX-1, we find that the specific genes required for secretion of EspB differ from those required for the secretion of CFP-10. Biochemical investigation demonstrates that EspB forms a complex with Rv3879c and that Rv3879c interacts with Rv3871, the same component of ESX-1 that interacts with the ESAT-6/CFP-10 complex during its secretion. These data support a model that different substrates are delivered to the ESX-1 machine by molecularly distinguishable pathways. Moreover, each of these pathways for ESX-1-mediated secretion contributes to mycobacterial virulence.

Results
Both M. tuberculosis and M. marinum EspB Restore Intracellular Growth to the M. marinum Mutant espB M ::tn A previous genetic screen for M. marinum mutants that fail to cause hemolysis led to the isolation of eight mutants in the extended RD1 (extRD1) locus, Mh3866-Mh3881c [7]. Of the eight mutants, espB M ::tn (Mh3881c::tn) was the most attenuated for virulence to zebrafish, growth in macrophages, and cytotoxicity to J774 cells. Thus, we decided to investigate the espB M -encoded protein (EspB M ) and its M. tuberculosis homolog (EspB T ) in detail. The gene, espB M , is the first in a two-gene operon. Using quantitative RT-PCR, we found that the mutation disrupts the expression of both genes in the operon (unpublished data). We then sought to determine the genetic requirements for restoration of intracellular growth to the mutant. Introduction of a non-integrating plasmid, expressing either EspB M from the espB M promoter or EspB T from its native promoter, was sufficient to appreciably restore growth in macrophages to espB M ::tn ( Figure 1A). The non-integrating plasmids expressing both EspB M and Mh3880c or both EspB T and Rv3880c were not superior in restoration of intracellular growth. Thus, EspB is necessary and sufficient to appreciably complement espB M ::tn, and the M. tuberculosis homolog functions equally well in M. marinum, demonstrating conservation of function.
While the expression of EspB M from a non-integrating plasmid appreciably restored growth in macrophages to espB M ::tn, the complementation was not complete. Among possible explanations are that the transposon insertion exerted a polar effect on the operon upstream, Rv3883c-Rv3882c, which also might have a role in intracellular growth, or that a proper stoichiometry between EspB and ESX-1 is required for complete complementation. Therefore, integrating plasmids encoding either espB M along with the espB M promoter, espB M -Mh3880c along with the espB M promoter, or the entire locus Mh3883c-Mh3880c along with the Mh3883c promoter, were introduced into espB M ::tn. The locus Mh3883c-Mh3880c, along with the Mh3883c promoter, was also introduced into espB M ::tn on a non-integrating plasmid. Of these constructs, only the integrating plasmids encoding espB M -Mh3880c or Mh3883c-Mh3880c fully complemented the growth defect of espB M ::tn ( Figure 1B). Similarly, espB M alone appreciably restored a rough colony morphology to the espB M ::tn mutant, but espB M -Mh3880c or Mh3883c-Mh3880c fully restored the rough colony morphology to espB M ::tn ( Figure S1). These results suggest that Mh3880c can contribute

Author Summary
A major mechanism used by pathogenic bacteria for disabling host defenses is secretion of virulence proteins. These effectors are often transported by specialized secretion machines. One such pathway, present in Mycobacterium and other Gram-positive genera, is ESX-1 (early secretory antigenic target 6 system 1). Although ESX-1 is required for multiple phenotypes related to the pathogenesis of infection, only three substrates of the secretion machine have been identified to date, and the mechanism by which these substrates are exported is not understood. In our efforts to understand this virulence-related secretion mechanism, we identified a novel substrate and found that its delivery to the ESX-1 machine requires different protein interactions than previously identified substrates. Finally, we present data that the various ESX-1 substrates contribute additively to virulence. These data are incorporated into a model of ESX-1 function.
to M. marinum growth in macrophages when it is expressed along with espB M from the bacterial chromosome. In contrast, espB M contributes equally well to bacterial virulence whether expressed episomally or on the chromosome, suggesting that its contribution is more independent of its stoichiometry with respect to other virulence components.

EspB Is a Secreted Protein That Undergoes Carboxyterminal Processing
As a first step toward understanding the role of EspB in virulence and growth in macrophages, we determined its localization in Mycobacteria grown in broth culture. The cell lysate and culture filtrate fractions of M. tuberculosis H37Rv, wild-type M. marinum, and M. marinum espB M ::tn were probed with a mouse polyclonal antibody raised against a 100 amino acid fragment of EspB T extending from amino acid 234 to 333 ( Figure 2A). EspB was detected in both the cell lysate and the culture filtrate fractions of M. tuberculosis, as well as in both the cell lysate and the culture filtrate fractions of wildtype M. marinum. EspB was not detected in either fraction of the espB M ::tn culture, verifying the specificity of the antibody. GroEL, a non-secreted bacterial cytoplasmic protein, was found exclusively in the cell lysate, demonstrating that EspB did not appear in the culture filtrate as a result of cell lysis.
The EspB in the cell lysate had an M r of 55 kDa on SDS-PAGE, while the EspB in the culture filtrate of both species ran at a slightly lower molecular weight. A lower molecular weight of EspB in the culture filtrate was also observed in a prior proteomic analysis of M. tuberculosis H37Rv proteins [21], in which EspB in the cell lysate was observed on a 2-D gel as a single spot with an apparent molecular weight of 55.6 kDa, while the EspB in the culture filtrate was observed as two spots with apparent molecular weights of 49.7 kDa and 48.4 kDa. Therefore, EspB might be cleaved either during or after secretion. To test this possibility, a V5 epitope tag was fused to the N-terminus of EspB M and a His6x epitope tag was fused to the C-terminus. The resulting construct, V5-EspB M -His6x, was expressed in the espB M ::tn mutant. Like the native protein, V5-tagged EspB was detected in the cell lysate as a single band and as a doublet in the culture filtrate ( Figure 2B). In contrast, His-tagged protein was only detected in the cell lysate fraction, suggesting that EspB M in the culture filtrate is Cterminally truncated.

EspB Secretion Requires a Distinct Set of extRD1-Encoded Genes for Secretion
To assess which ESX-1 genes are required for EspB secretion, its compartmentalization between cell lysate and culture filtrate was determined for several M. marinum ESX-1 mutants ( Figure 2C). Although EspB M was found in both the cell lysate and culture filtrate fractions of most mutants, EspB M was not detected in the culture filtrates of MmDRD1, Mh3868::tn, Mh3879c::tn, or Mh3871::tn.
The Mh3868::tn mutants failed to accumulate protein in the pellet, suggesting that Mh3868 protein could be involved in EspB synthesis or stability. Thus, of the ESX-1 genes tested, only Mh3879 and Mh3871 were clearly involved in EspB M secretion. In contrast, none of the mutants secreted ESAT-6 [7], and only Mh3879c::tn and Mh3878c::tn secreted CFP-10 normally. This difference in secretion requirements for ESAT-6 and CFP-10 in M. marinum has been noted previously [7]. Complementation of Mh3879::tn and espB::tn restored EspB M secretion. GroEL was absent from culture filtrates of all strains, and secretion of the fibronectin attachment protein (FAP), a protein secreted in a Sec-dependent manner [22], was not disturbed in any of the extRD1 mutants. Thus, the product of the espB M gene is a secreted protein that requires Mh3871, a core component of the ESX-1 secretion machine, for export; we have therefore named it ESX-1 substrate protein B (EspB). However, EspB, ESAT-6, and CFP-10 differ with respect to the extRD1 genes required for their secretion. EspB M secretion depends on Mh3879c, but is independent of Mh3866 and Mh3867, while CFP-10 shows the inverse pattern.
To demonstrate the importance of the ESX-1 machine in EspB secretion in another strain of M. marinum, we examined the 1218R strain and an isogenic mutant in which the Mh3871 gene had been disrupted. The M strain, used for the previous experiments, is a human isolate, whereas 1218R was originally isolated from an infected fish. Wild-type 1218R secreted EspB, but the Mh3871 mutant did not ( Figure S2  To test directly whether ESX-1 was required for EspB secretion by M. tuberculosis, we examined culture filtrates from M. tuberculosis Erdman and the isogenic mutants Rv3870::tn, Rv3871::tn, and DCFP-10 ( Figure 2D). Secretion of EspB T by wild-type M. tuberculosis was abrogated in the Rv3870 and Rv3871 mutants, but not in the DCFP-10 mutant. Thus, EspB is a secreted protein in both M. marinum and M. tuberculosis, and its secretion requires core ESX-1 components in both species of Mycobacteria. Importantly, EspB is the first ESX-1 substrate in M. tuberculosis whose secretion is not disrupted in the DCFP-10 mutant.
MmDRD1, espB M ::tn, and Mh3871::tn Are More Attenuated for Growth in Macrophages than the Other M. marinum extRD1 Mutants Of the ten M. marinum extRD1 mutants we examined, MmDRD1, espB M ::tn, and Mh3871::tn were disrupted for the secretion of all three substrates: ESAT-6, CFP-10, and EspB M . In contrast, the Mh3879::tn mutant was disrupted only for the secretion of ESAT-6 and EspB M , while the Mh3866::tn and Mh3867::tn mutants were disrupted only for ESAT-6 and CFP-10 secretion. To assess the importance of the multiple ESX-1 substrates for growth in macrophages, we infected murine bone marrow-derived macrophages (BMDMs) with wild-type M. marinum, with strains lacking one secreted effector, or with strains lacking secretion of all the known ESX-1 substrates. As shown in Figure 3, MmDRD1, espB M ::tn, and Mh3871::tn, which fail to secrete all substrates, are more attenuated for growth in macrophages than Mh3866::tn, which still secretes EspB M , or Mh3879c::tn, which still secretes CFP-10. Therefore, we conclude that the various substrates of ESX-1 each contribute to virulence.

EspB Physically Interacts with the Rv3879c
To learn more about the involvement of ESX-1 in EspB secretion, we tested whether EspB would interact with other ESX-1 genes by bacterial two-hybrid analysis ( Figure 4). An advantage of the bacterial two-hybrid system is that it can allow detection of interactions of membrane-bound proteins [23]. In this assay, potential protein-protein interactions are assessed by determining the ratio of colonies that grow on selective medium to the number grown on non-selective medium. For each of the bait plasmids, co-transformation with an empty target resulted in a ratio of colonies on selective to non-selective medium of less than 0.1%, as did cotransformation of the EspB T target with an empty bait. In contrast, the Rv3879c bait and EspB T target resulted in a ratio of 7.6%, an increase of more than 75-fold. An Rv3876 bait also showed interaction above background with EspB T , but since the M. marinum Mh3876::tn mutant showed significant EspB M secretion ( Figure 2C), any interaction between Rv3876 and EspB T is not likely to be required for EspB secretion and thus was not pursued.
To test for an analogous interaction between EspB M and Mh3879c and to confirm the potential interaction between EspB T and Rv3879c suggested by the two-hybrid assay, we performed in vitro pull-down assays. All of the proteins used were expressed in Escherichia coli as GST-or V5-epitopetagged fusions. Controls for nonspecific interactions included GST alone, as well as GST-syntaxin2, and GST-Shp1. As shown in Figure 5A, GST-tagged EspB M , but none of the GST controls, bound specifically to V5-tagged Mh3879c. In the reciprocal experiment, GST-tagged Mh3879c bound specifically to V5-EspB M . Similarly, as shown in Figure 5B, GSTtagged EspB T bound specifically to V5-tagged Rv3879c, and GST-tagged Rv3879c bound specifically to V5-tagged EspB T . These data demonstrate that recombinant EspB T and Rv3879c, as well as their M. marinum homologs, interact in vitro.
Since Rv3871 mutants in both M. tuberculosis and M. marinum fail to secrete EspB, we used GST pulldowns to test whether Rv3871 interacts with either EspB T or Rv3879c. GSTtagged Rv3879c bound to V5-tagged Rv3871, whereas the GST controls and GST-EspB T did not bind to Rv3871. This suggests that Rv3879c may facilitate EspB T secretion through an interaction with Rv3871.

The Carboxyterminus of EspB Is Dispensable for Interaction with Mh3879c and for Secretion
To identify whether EspB, like CFP-10, requires its carboxyterminus for secretion, we constructed a series of EspB M deletion mutants with N-terminal V5 tags and expressed them in the espB M ::tn mutant strain using the espB M promoter. As shown in Figure 6A, V5-tagged full-length EspB M was secreted. This N-terminally tagged protein, like native EspB M , underwent C-terminal truncation either during or after secretion. EspB M deletion mutant constructs D(2-31), D(264-271), and D(400-454) were stably expressed in M. marinum, but only EspB M D(400-454) accumulated in the culture filtrate. The secreted EspB M D(400-454) had a higher apparent molecular weight than the secreted full-length EspB M , presumably because deletion of the C-terminal 55 amino acids inhibits some of the carboxyterminal proteolytic processing. This result demonstrates that the C-terminus of EspB M is dispensable for secretion, but N-terminal and internal amino acids are required. Next, we tested how these EspB M mutants interacted with Mh3879c. Lysates of E. coli that express V5-tagged EspB M mutants were incubated with GST-Mh3879c. While full-length EspB M and EspB M D(400-454) bound to GST-Mh3879c, the stably expressed but nonsecreted EspB M D(2-31) and EspB M D(264-271) constructs did not bind to GST-Mh3879c ( Figure 6B). These data support a model in which EspB interacts with Rv3879c, which in turn interacts with Rv3871, to facilitate the secretion of EspB.  Figure S3A). In wild-type M. marinum, V5-Mh3879c and V5-EspB M were expressed at nearly identical levels in the cell lysate, but only V5-EspB M was detected in the culture filtrate ( Figure S3A). To determine whether failure of secretion reflected inefficient competition of V5-tagged protein with native protein, the V5-EspB M secretion was also analyzed in the Mh3879::tn mutant. In this strain as well, V5-Mh3879c was found only in the cell lysate. Thus, V5-tagged Mh3879c, while fully competent to mediate EspB secretion, was not itself secreted, suggesting that Mh3879c and EspB are not secreted as a heterodimer. The C-terminally His6x-tagged Mh3879c, which did not restore EspB secretion to the Mh3879::tn mutant, also was detected only in the cell lysate. Since Mh3879c-His6x failed to complement the EspB M secretion defect of the Mh3879c::tn mutant, we hypothesized that the carboxyterminus of Rv3879c might be required for interaction with EspB. To test this hypothesis, lysates of E. coli that express V5-tagged Rv3879 mutants were incubated with GST alone, GST-Rv3871, or GST-EspB T . While full-length Rv3879 and Rv3879 D(1-166) bound to GST-EspB T , Rv3879 D(564-729) failed to bind to GST-EspB T ( Figure S3B). None of the constructs bound to GST alone, and all three constructs bound to GST-Rv3871. Thus, the carboxyterminal 166 amino acids of Rv3879 are required for EspB secretion, but not for interaction with the ESX-1 machine.

Discussion
In this study, we identified EspB as a novel substrate of the ESX-1 secretion system and demonstrated a requirement for the Mh3879c and Mh3871 genes in the secretion of EspB M. Further, we showed protein complex formation between EspB M and Mh3879c, as well as identical behavior of their M. tuberculosis homologs. Two mutants of EspB M that were stable after synthesis but failed to bind Mh3879c were not secreted, while a large carboxylterminal deletion did not interfere with either Mh3879c binding or secretion. Additionally, the carboxyterminus of Rv3879c/Mh3879c is required for interaction with and secretion of EspB. These results suggest that the EspB/Mh3879c protein complex is required for EspB M secretion. While complex formation between ESAT-6 and CFP-10 is required for their secretion as a heterodimer by M. tuberculosis, Mh3879c appears not to be secreted. Our data, though, do not exclude the possibility that the aminoterminus of Mh3879c is quantitatively removed during or immediately after secretion, since we do not have and could not probe with antibodies to the native protein. We hypothesize that Mh3879c acts as a cytosolic chaperone to deliver EspB M to the secretion machine. We showed that Rv3879c interacts directly with Rv3871 and that Rv3871, in addition to being required for the secretion of ESAT-6/CFP-10, is required for the secretion of EspB. Although our work does not reveal precisely how EspB is delivered to the ESX-1 machine, our data demonstrate that Rv3879c can interact with Rv3871 as well as with EspB T , suggesting that EspB may be targeted to Rv3871 in this way. We propose that the The target plasmid containing EspB T fused to the RNA polymerase alpha subunit was co-transformed with each of the bait plasmids containing the indicated extRD1 proteins fused to the lambda repressor into the reporter validation strain. Shown is the ratio of growth of the co-transformants on selective (þ5 mM 3AT) versus non-selective plates. The experiment depicted is representative of three independent determinations. doi:10.1371/journal.ppat.0030105.g004 mechanisms of EspB and CFP-10 secretion intersect at binding to Rv3871 (Figure 7).
We also found that disruption of Mh3868 leads to loss of accumulation of EspB in the bacterial cytosol. We previously observed that disruption of Mh3868 prevents bacterial accumulation of ESAT-6 and CFP-10 [7]. Mh3868 and its M. tuberculosis homolog Rv3868 are predicted to be AAA ATPases, which suggests that they may function as chaperones for the translocation of ESX-1 substrates, but little is known about this key protein. We have found that CFP-10 and espB M mRNAs are expressed in the Mh3868::tn mutants (B. McLaughlin and E. Brown, unpublished data), suggesting that the Mh3868 gene product affects either the translation or stability of the ESX-1 substrates. Characterizing the function of Mh3868 will certainly be important to better understand ESX-1-mediated secretion.
Like ESAT-6 and CFP-10, EspA is secreted by the ESX-1 machine. Whether any of the M. marinum genes with sequence similarity to espA are functional orthologs has not yet been determined. Loss of either EspA or EspB inhibits secretion of ESAT-6 and CFP-10, but the reason for their requirement is unknown. It may be that as substrates reach the final common pathway for secretion, they interact in a manner that leads to cooperative secretion. Clearly, though, the secretion of EspB is quite distinct from that of EspA. While EspA requires CFP-10 for its secretion, EspB secretion is independent of CFP-10. EspB secretion is not disrupted in the M. marinum mutants Mh3866::tn and Mh3867::tn, neither of which secrete CFP-10, nor is EspB secretion disrupted in the M. tuberculosis DCFP-10 mutant. These data are consistent with the model that EspB, unlike either ESAT-6 or EspA, is targeted to the ESX-1 machine independently of CFP-10.
These studies beg the question of whether it is possible to determine which ESX-1 substrates are most important for virulence. This has been a difficult task because of the apparent codependence of the various substrates on each other for secretion. However, our results allowed a somewhat different approach. We used a set of extRD1 mutants in which some (Mh3866::tn and Mh3867::tn) failed to secrete CFP-10, but did secrete EspB; while another mutant (Mh3879::tn) secreted CFP-10 but failed to secrete EspB; while mutants that disrupted the core secretion machinery (MmDRD1 and  Mh3871::tn) and espB M ::tn itself failed to secrete all substrates. We found that mutants lacking secretion of both substrates had a more marked growth defect in macrophages than the mutants lacking secretion of only one substrate. This suggests that the different substrates make distinct, and potentially additive, contributions to virulence. Although we cannot say that the defects in intracellular growth of the various mutants are caused by the substrates we have identified, our work does support the hypothesis that ESX-1 secretes more than one substrate that contributes to the virulence of Mycobacteria and that different substrates may have independent contributions to bacterial pathogenesis.
In summary, this work has identified a novel substrate for ESX-1-dependent secretion and has demonstrated interactions of this substrate with a protein encoded within RD1, expanding our understanding of how genes within this locus contribute to this novel secretion pathway. Furthermore, we have demonstrated that secretion of distinct ESX-1 substrates follows variable pathways to interaction with the core secretion machinery, and that the different substrates may contribute independently to intracellular survival and growth of the bacteria. These data extend the understanding of a major virulence mechanism of Mycobacteria.

Materials and Methods
Bacterial strains and plasmids. All strains and plasmids used in this study are listed in Table 1. M. marinum strains were grown as previously described [24] To construct the plasmids pBM841, pBM540, and pBM810, the genes Rv3871, Rv3879c, and Rv3881c were PCR amplified from the cosmid RD1-2F9 [25] and ligated into pBM510, a derivative of pET22bþ in which the N-terminal His tag was replaced with the V5 epitope tag. To construct the plasmids pBM843 and pBM504, the genes Mh3879c and Mh3881c were PCR amplified from M. marinum M genomic DNA and ligated into pBM510. To construct the plasmids pBM332 and pBM336, a series of fragments were ligated into pLYG206 to achieve the following sequence ligated into the NotI and XbaI sites: 250 bp upstream of espB M , then the V5 epitope, then the espB M gene, and finally, in the case of the pBM336 plasmid, the His6x epitope. The plasmids pBM869, pBM870, and pBM871 were made in a manner synonymous to that of pBM332 and pBM336, where the Mh3879 promoter and gene were used. The plasmids pBM367 and pBM400v were constructed by PCR from pBM332 and re-ligation of the truncated gene fragments back into pBM332, while pBM398 was generated by quick-change mutagenesis (Stratagene, http://www.stratagene.com/). For pBM589, pBM398e, and pBM400ve, the espB M gene fragments in the plasmids pBM367, pBM398, and pBM400v were cut by restriction digest and ligated into pBM504. For pBM856, pBM550, pBM553, and pBM551, the genes Mh3879c, espB M , Rv3879c, and espB T were cut by restriction digest from the plasmids pBM843, pBM504, pBM540, and pBM810 and ligated into the GST expression vector pGex-KG. To construct the plasmid pMh3879, 250 bp upstream of the gene Mh3879c together with Mh3879c was PCR amplified from M. marinum genomic DNA and inserted into pLYG206. The plasmids pBM1010 and pBM1013 were constructed by restriction digests of pBM540 to excise portions of Rv3879c, and  ligation of 59 phosphorylated hybridized oligos that restored the frame and created the Rv3879 deletions D(1-166) and D(564-729). To construct the plasmid p(Mh3883c-Mh3880c), the 345 bp upstream of Mh3883c along with Mh3883c-Mh3880c was cut by restriction digest from the plasmid pBM263 and inserted into pLYG206. To construct the plasmid p(espB T -Rv3880c), 250 bp upstream of the operon Rv3881c-Rv3880c together with the operon were inserted into pLYG206.
Protein preparation and analysis. M. marinum strains were grown in 40-mL cultures to 0.5 OD 600 in 7H9 medium. The cultures were centrifuged and washed three times with 15 mL of PBS before resuspension in 40 mL of Sauton's medium, supplemented with 0.015% Tween-80. When strains containing non-integrating plasmids for complementation were grown in Sauton's medium, the Sauton's medium was supplemented with Zeocin (5 lg/ml; Invitrogen, http:// www.invitrogen.com/). After growth for 36 h at 30 8C, 105 rpm, in Sauton's medium, the cells were harvested by centrifugation. Supernatants were filtered through a 0.22-lm-pore-size filter with a glass pre-filter and concentrated with an Amicon Ultra-15 (5,000-molecular-weight cutoff; Millipore, http://www.millipore.com/) to 200 lL, which was saved as the culture filtrate (CF) fraction.
Pelleted cells were washed and resuspended in 1.5 mL of PBS with a protease inhibitor cocktail and 1 mM PMSF. Pellets were lysed using glass beads and the mini-bead beater (BioSpec Products, http://www. biospec.com/) with three 40-s pulses at maximum speed and incubations on ice in between each pulse, and then centrifuged at 3,000g for 2 min at 4 8C to remove unbroken cells. The resulting supernatant was collected and saved as the cell lysate (CL) fraction. M. tuberculosis (Erdman) culture filtrate and cell lysate fractions were prepared as previously described [1]. Total protein concentrations were determined by a Bradford assay.
Bacterial two-hybrid system assay. The genes Rv3614c, Rv3615c, and Rv3616c, which were PCR amplified from genomic DNA, and each of the genes in the region Rv3864 through Rv3883, which were PCR amplified from the cosmid RD1-2F9 [25], were cloned into the ''bait'' vector pBT (BacterioMatch II; Stratagene) in frame with cI. Rv3881c was cloned into the ''target'' vector pTRG in frame with the N-terminal subunit of RNA polymerase according to the manufacturer's instructions. The constructs were co-transformed into the E. coli two-hybrid system reporter validation strain XL1-Blue MRF9 hisB lac [F9 laqIq HIS3 aadA Kanr] and plated onto both the selective (þ5 mM 3AT) and the non-selective screening medium according to the manufacturer's instructions. The non-selective screening plate is histidine-dropout M9 agar supplemented with 0.5 mM IPTG, 12.5 lg/ ml tetracycline, and 25 lg/ml chloramphenicol. The selective screening plate is histidine-dropout M9 agar supplemented with 0.5 mM IPTG, 12.5 lg/ml tetracycline, 25 lg/ml chloramphenicol, and 5 mM 3amino-1,2,4-triazole.
GST pulldown. GST fusion proteins, GST alone, and V5-tagged proteins were expressed in the BL21-RP codon plus E. coli strain (Stratagene) by addition of 0.2 mM IPTG (3 h at 30 8C). Bacterial cultures were lysed in buffer containing 50 mM HEPES (pH 7.4), 300 mM NaCl, 1% Triton X-100, 0.5 mM EDTA, and protease inhibitor cocktail (Roche). Solubilized proteins were separated by centrifugation at 20,000g for 10 min. The GST fusion proteins and GST alone were bound to glutathione agarose beads (Amersham Biosciences, http://www.gelifesciences.com) by incubation overnight at 4 8C. The beads were then extensively washed with PBS containing 0.1% Triton X-100. Bacterial lysates containing solubilized V5-tagged proteins, in lysis buffer, were incubated with the GST protein-loaded agarose beads overnight at 4 8C. After washing three times with PBS containing 1% Triton X-100, bead-bound protein was eluted in Laemmli buffer, seperated by SDS-PAGE, and analyzed by western blot.
Macrophage infections. All macrophages used in these experiments were derived from bone marrow cells of C57BL/6 mice that were differentiated for 6 d in DMEM supplemented with 10% CMG supernatant [26] and 10% fetal bovine serum (FBS; HyClone, http:// www.hyclone.com/). Immediately prior to infection, macrophage monolayers were washed once with FBS-free DMEM. M. marinum strains were each grown to OD 600 of 1.0, prepared for infection, and incubated with macrophages as previously described [7]. All infections were performed at a multiplicity of infection of 1, for 2 h at 32 8C, in a 5% CO2, humidified environment, in 24-well plates. The time at which M. marinum was added to the well was designated time zero. At the end of the 2 h incubation period (T ¼ 2 h), infected monolayers were washed twice with DMEM and further incubated in DMEM containing 0.1% FBS and 200 lg of amikacin/ml for 2 h to kill extracellular bacteria. At the end of the antibiotic treatment, monolayers were washed twice with DMEM and incubated in DMEM containing 0.1% FBS at 32 8C and 5% CO2. Intracellular bacteria were enumerated by lysing macrophage monolayers and diluting and plating bacteria exactly as described [7]. Statistical analysis was performed by calculating the one-way analysis of variance (ANOVA) with GraphPad Prism 4.0 (GraphPad Software, http://www.graphpad. com). Figure S1. Colony Morphology of espB M ::tn Dilutions of each strain were grown on 7H10 agar, without antibiotics, and imaged after 12 d. Found at doi:10.1371/journal.ppat.0030105.sg001 (5.9 MB TIF). Figure S2. Mh3871 Is Required for the Secretion of EspB M Cell lysates (CL) and culture filtrates (CF) were prepared from wildtype M. marinum strain 1218 and isogenic mutants as described in Materials and Methods. CL and CF proteins were separated by SDS-PAGE and the indicated proteins detected by western blot. Cultures of each strain were inoculated into Sauton's medium at an optical density (OD) of 0.5 and grown for 36 h. Therefore, the samples of each strain are normalized by OD readings. Of the total CL and CF fractions collected for each strain from one experiment, 3% of the CL was loaded in each lane and 15% of the total CF was loaded into each lane. Found at doi:10.1371/journal.ppat.0030105.sg002 (1.0 MB TIF). Figure S3. Mh3879c Is Not Secreted and the Carboxyterminus of Rv3879 Is Required for Interaction with EspB Cell lysates (CL) and culture filtrates (CF) were prepared from the indicated strains as described in Materials and Methods. Proteins were separated by SDS-PAGE and the indicated proteins were detected by western blot strains as described in Materials and Methods. (A) Cultures of each strain were inoculated into Sauton's medium at an OD of 0.5 and grown for 36 h. Therefore, the samples of each strain are normalized by OD readings. Of the total CL and CF fractions collected for each strain from one experiment, 3% of the CL was loaded in each lane and 15% of the total CF was loaded into each lane. (B) Agarose beads with immobilized GST only, GST-Rv3871, or GST-EspB T were incubated with E. coli lysates that express V5-tagged Rv3879c full-length (1-729), Rv3879c D(1-166), or Rv3879c D(564-729). The input lysate (0.1%) and the material from the cell lysates that bound to the beads was run on SDS-PAGE and detected by western blotting with an antibody against V5. Found at doi:10.1371/journal.ppat.0030105.sg003 (2.9 MB TIF).

Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers for the gene products discussed in this paper are