Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

High throughput identification of Mtb secretome-human host interactions

We used a systematic strategy to identify secreted bacterial virulence factors by looking for interactions with host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform [22]. First, we curated the literature to define the Mtb secretome. Thirty-eight publications predicted 718 secreted proteins based on presence in culture filtrate (CF), ability to cause secretion of an assayable protein, bioinformatic criteria, or detailed study (see Text S1 for additional details). In order to prioritize open reading frames (ORFs) for screening, we imposed a number of criteria, such as excluding proteins with multiple transmembrane spanning domains (see Text S1 for additional details). In addition, since the starting list of putative secreted proteins might contain proteins that are not actually secreted, we attempted to eliminate ORFs that were likely to be inaccurately classified as secreted. One way in which this can happen is if cytoplasmic proteins appear in CF due to bacterial lysis. In order to minimize the contribution of such proteins, we did not include ORFs that were annotated in Tuberculist (http://tuberculist.epfl.ch/) as being involved in lipid metabolism, information pathways (which contains proteins involved in replication, transcription and translation), or intermediary metabolism and respiration, since most of these are likely involved in basic, intrinsic bacterial processes, and hence, many may be misclassified. To avoid removing true secreted proteins, ORFs were not de-prioritized if they had a possible signal sequence or there were data supporting their role during infection. In doing so, we removed many proteins that were found in CF in a single study, and hence may be misclassified (see Text S1 for details). From the final list, 339 sequence validated secretome ORFs were provided by Pathogen Functional Genomic Resource Center (PFGRC; Dataset S1). Because many secreted proteins play an intrinsic role in the bacterial lifecycle, we anticipated that only a small fraction would interact with human proteins. Thus, to estimate a false positive hit rate of our system, we included sixty ORFs that are not likely to be secreted to serve as controls (see Text S1 for details; Dataset S2).

In order to evaluate their performance in Y2H protein-protein interaction (PPI) mapping, we tested the 399 Mtb ORFs expressed as Gal4-DNA binding domain (DB) fusions for pair wise interactions with the same 399 Mtb ORFs expressed as Gal4-activation domain (AD) fusions. From the ∼160,000 combinations queried, we found 14 unique PPIs (Table S1). The rate of interactions is as high as in human ORFeome mapping [22], exceeding the stochastic false positive rate of the Y2H platform by fifteen-fold [23]. Half of the interactions were between proteins belonging to the WXG100 family (EsxA-EsxW). These proteins are approximately 100 amino acids in length and have a characteristic hairpin bend formed by a Trp-Xaa-Gly (W-X-G) motif. Mtb encodes 11 tandem pairs of such proteins, which are thought to function as secreted heterodimers. Heterodimer formation is proposed to be limited to interactions between genome pairs or very closely related family members [24], [25], and the interactions we detected by Y2H exhibit this specificity. Six of the remaining seven PPIs involved homotypic interactions; for example, bacterioferritin (BfrB) was found to interact with itself, consistent with the proposal that it assembles into 24-subunit oligomers [26].

After ensuring the high performance of Mtb ORFs in Y2H PPI mapping, we looked for interactions between the Mtb secretome and ∼12,000 human ORFs, testing approximately 4 million interactions. From the secretome collection, we identified 99 PPIs between 53 Mtb proteins and 63 human proteins (Dataset S3). The number of Mtb proteins exhibiting an interaction with a human protein was approximately two-fold higher for the secretome collection compared to the non-secreted control set (53 out of 339 versus 5 out of 60). We analyzed the collection to determine whether PPIs were enriched for subsets of Mtb proteins (Table S2). We observed that the sixteen Esx proteins included in the collection were significantly more likely to interact with human ORFs than were controls (p = 0.0087). The finding that Esx proteins were enriched for interactions may reflect that this group of proteins plays an important role in virulence, or could mean that these proteins, which usually form a heterodimer, are prone to aberrant interactions when they are expressed without their binding partner.

It is difficult to gauge the success of the screen based upon known interaction between Mtb proteins and cytosolic human protein because so few are known. Included in our screening set were, PtpA, which has been shown to interact with Vps33B and the H subunit of the human v-ATPase [27], [28], LpdC, which interacts with coronin 1 [29], and NdkA, which interacts with Rab5 and Rab7 [30]. We did not identify these known interactions, however, the screen was not performed to saturation and the Y2H platform can detect ∼20% of well-validated interacting pairs [31]. We did identify an interaction between PtpA and Ligand of Numb protein X (LNX1), a RING finger-type E3 ligase that contains 4 PDZ domains and plays a scaffolding role in diverse cellular pathways. Several other secretome ORFs also interacted with LNX1, suggesting that LNX1 might be modulated by Mtb, or LNX1 might regulate the function or stability of certain Mtb effectors.

When we examined hit rates based upon the functional category of the Mtb ORFs, the category with the greatest enrichment was cell wall and cell processes, which contains the Esx family members. The intermediary metabolism and respiration category exhibited a hit rate similar to the control collection, consistent with the idea that most of these ORFs do not function in host interactions as their annotation suggests. Interestingly, one of the two proteins that did exhibit an interaction in this category is Zmp1, a zinc metalloprotease which has been shown to inhibit the inflammasome and impair phagosome maturation but whose cellular target is unknown [32]. Zmp1 interacted with KCTD6, a BTB/POZ domain containing protein that can function as a Cullin3 (Cul3) adaptor [33]. Cul3 has recently been shown to be a regulator of endo-lysosomal trafficking, suggesting that Zmp1 may impair phagosome maturation by acting on KCTD6-Cul3 [34].

To evaluate the human targets of the Mtb proteins, we searched the STRING database (http://string.embl.de/) for each of the proteins' functional associations. The STRING database predicts protein-protein interactions based upon physical and functional associations, such as available high-throughput data, co-expression, genomic context, and text mining of available literature. Using a medium confidence value to define protein-protein interactions, there were significantly more interactions (n = 16) observed for the human targets than would be predicted by chance (p = 8.3×10⁻¹²). We identified proteins involved in host immunity to bacterial infection, such as Ndp52 [35], [36], Tax1pb1 [37], and STAT3 [38], however, human targets of the Mtb proteins were not significantly enriched for annotated pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) when corrected for multiple testing, which may reflect the limited number of human targets found.

ESCRT is required for restricting intracellular growth and trafficking of slow growing mycobacteria

We focused on the interaction between EsxH and Hrs because TSSSs, which secrete Esx proteins, are clearly important in virulence but the function of their secreted effectors is largely unknown. In addition, our existing data supported the idea that the ESCRT machinery is important in controlling bacterial replication. Hrs, which plays a central role in the assembly of the initial ESCRT components on endosomes, is recruited to mycobacterial phagosomes [39], and we had previously shown in an RNAi screen in Drosophila that ESCRT restricts the intracellular growth of rapidly growing mycobacteria [40], [41]. Control of bacterial replication appears to be particularly sensitive to ESCRT perturbation, because, in addition, when we screened ∼6500 siRNA pools in RAW 264.7 (RAW) macrophages for their ability to confer enhanced intracellular growth of Msmeg, we found that the two strongest hits were Rab7, known to be involved in late endosome-lysosome fusion, and Tsg101, an ESCRT-I component that is recruited to endosomes by Hrs (data not shown). Hrs was also identified in this screen, although previously we had found no effect with Hrs silencing, which we now attribute to insufficient protein depletion [41]. In the RAW cell RNAi screen that identified Hrs, we used Ambion Silencer siRNA pools, whereas previously we used a Dharmacon siGENOME pool to deplete Hrs [41]. To clarify the discrepancy, we tested a third pool (Dharmacon ON-TARGETplus), which, like the Ambion pool, conferred enhanced growth to Msmeg. We tested the individual Dharmacon ON-TARGETplus siRNAs and found that 2 of 4 targeting Hrs resulted in depletion of Hrs protein, enhanced the growth of Msmeg, and altered trafficking, whereas the other two had no effect (Figure S1 and data not shown). Thus, one possibility is that Mtb secretes EsxH, which binds Hrs and impairs ESCRT function, thereby promoting intracellular bacterial growth. To determine whether ESCRT restricts growth of Mtb, we depleted Hrs and Tsg101 and examined the intracellular growth of Mtb in RAW macrophages. We found no significant effect of silencing on bacterial uptake (data not shown), however when we assessed bacterial colony forming units (CFU) two day post-infection, we observed enhanced intracellular survival of Mtb in cells depleted of Hrs or Tsg101, similar to what was seen with Rab7 silencing (Figure 1A). Intracellular growth of BCG in bone marrow-derived macrophages (BMDMs) was even more strongly effected (Figure S2). Thus, Hrs restricts growth of slow growing and virulent mycobacteria.

ESCRT targets certain cell surface receptors and biosynthetic cargo to lysosomes [42]. Thus, ESCRT might restrict intracellular bacterial growth by governing bacterial trafficking and/or lysosomal content. We examined the localization of Mtb relative to Transferrin Receptor (TfR), a marker of early and recycling endosomes, and LAMP1, a marker of late endosomes and lysosomes using automated image analysis (Figures S3A, S3B). In cells depleted of Tsg101, Hrs, or Rab7, we observed diminished co-localization between Mtb and LAMP1 and a concomitant increase in co-localization of Mtb with TfR compared to control cells 24 hours post-infection (hpi) (Figure 1B), suggesting decreased Mtb delivery to degradative compartments. Similarly, in cells infected with BCG there was diminished co-localization with LysoTracker, which accumulates in the acidic environment of the lysosome, and enhanced co-localization with TfR (Figure 1B). Thus, Hrs and Tsg101, like Rab7, are required for bacterial trafficking. To verify that bacterial viability correlates with low LAMP1 and LysoTracker co-localization and with high TfR co-localization, we compared the cellular localization of viable bacteria to total bacteria. We identified metabolically active BCG 24 hpi based upon their ability to induce expression of GFP from a tetracycline-inducible promoter (BCG-tet-GFP) and compared their intracellular localization to the BCG strain that constitutively express GFP (BCG-GFP). Whereas there was a wide range in intensities of associated LAMP1 and LysoTracker with BCG-GFP, metabolically active bacteria were found almost exclusively in phagosomes with minimal acidification, little co-localization with LAMP1, and enhanced TfR co-localization at 48 hpi (Figure S3C). Thus, impaired bacterial trafficking to a late endosomal or lysosomal compartment underlies the failure to control mycobacterial replication in ESCRT-depleted cells, although altered lysosomal content may also contribute.

The Mtb EsxG EsxH heterodimer binds Hrs

Pathogenic mycobacteria arrest phagosome maturation in evolutionarily diverse cells. Supporting the idea that EsxH might play a role in inhibition of bacterial degradation, we observed that EsxH interacts with human, mouse, and zebrafish orthologs of Hrs, suggesting that it recognizes a conserved structural feature of Hrs (Figure 2A). Orthologs of EsxH are found widely in mycobacteria, including in the non-pathogen Msmeg. If EsxH prevents phagosome-lysosome fusion by impairing Hrs function, we anticipate that would be a feature specific to EsxH from pathogenic mycobacteria. To test this, we cloned the EsxH ortholog from Msmeg (MSMEG_0621; EsxH_Ms), which encodes a protein 75% identical to EsxH from Mtb (hereafter referred to as EsxH_Mt). Although EsxH_Ms interacted with EsxG_Mt, demonstrating that the protein was functional in the Y2H assay, it interacted poorly with Hrs (Figure 2A), consistent with the notion that the interaction of EsxH_Mt with Hrs contributes to virulence.

EsxH_Mt forms a heterodimer with EsxG_Mt, composed of a four-helix bundle with flexible N- and C-terminal arms from both proteins that coordinate zinc and contribute to a cleft that has been predicted to mediate a PPI [43]. To determine whether Hrs interacts with the heterodimer, we used a fusion protein in which EsxG_Mt and EsxH_Mt were expressed as a single polypeptide that preserves the folded structure of the native heterodimer [44]. This fusion protein interacted with Hrs (Figure 2B). Deletion of the first five amino acids of EsxH_Mt (EsxG_Mt-EsxH_Mt-ΔN5) weakened its interaction with Hrs (Figure 2B). These data show that Hrs can interact with EsxH_Mt when it is complexed to EsxG_Mt and suggest that the conformation of the amino terminal arm of EsxH_Mt is important. To further test whether the structure of the N- and C-termini are important, we mutated His-14, His-70, and His-76 Glu-77. These residues contribute to zinc binding, and His-76 is also part of the predicted cleft. We mutated them to Ala, with the exception of His-70, which we changed to Arg because this is found in EsxH_Ms. While H14A and H70R did not have a detectable effect, when His-76 and Glu-77 were both changed to Ala, the interaction between EsxH_Mt and Hrs was impaired, although EsxH_Mt H76A-E77A still interacted with EsxG_Mt (Figure 2A). To verify that EsxH_Mt binds Hrs, we purified the EsxG_Mt EsxH_Mt heterodimer from E. coli [44] and Hrs from baculovirus [45]. Hrs bound EsxG_Mt EsxH_Mt in a saturable manner, exhibiting stoichiometric binding with a Kd of ∼5 µM (Figure 2C and 2D). We conclude that Hrs interacts with the EsxG_Mt EsxH_Mt heterodimer, and the interaction likely involves the N- and C-terminal arms of EsxH_Mt.

EsxG_Mt and EsxH_Mt disrupt ESCRT function in mammalian cells

To determine whether EsxH_Mt interacts with Hrs and alters ESCRT function in mammalian cells, we expressed EsxH_Mt–FLAG in HEK293 cells. EsxH_Mt was not detectable unless we co-expressed EsxG_Mt. (Figure 3A, compare lanes 1 and 3; see Figure S4 for quantification); its abundance was also increased slightly by overexpression of Hrs (compare lane 1′ with 2′ and lane 5 with 6). When expressed alone, EsxH_Mt could be stabilized by MG132, likely because it is not properly folded without EsxG_Mt and hence is subject to proteasome-mediated degradation (Figure 3A compare lanes 1 and 5). To determine if there was an interaction between EsxG_Mt-EsxH_Mt and Hrs, we performed co-immunoprecipitation experiments in cells co-transfected with EsxG_Mt, EsxH_Mt, and Hrs-myc. Hrs was immunoprecipitated with an antibody directed against the myc-tag, and we found that EsxH_Mt was co-immunoprecipitated (Figure 3B). No EsxH_Mt was co-immunoprecipitated when an isotype control antibody was used, and as expected, EsxH_Ms and EsxH_Mt-H76A E77A were impaired in the interaction with Hrs (Figure 3B and 3C). Interestingly, the co-immunoprecipitation of EsxH_Mt and Hrs could only be detected when cells were pre-treated with MG132. Thus, one possibility is that the EsxG_Mt-EsxH_Mt heterodimer is polyubiquitinated and degraded by the proteasome. In the presence of MG132, the polyubiquitinated species might accumulate, allowing us to detect an interaction between Hrs and an ubiquitinated species of EsxH_Mt, since Hrs contains an ubiquitin interacting motif (UIM) domain. Arguing against this possibility, when EsxG_Mt was co-expressed with EsxH_Mt, there was little, if any, effect of MG132 on EsxH_Mt protein levels (Figure 3A, compare lanes 3 and 7, Figure S4, and Figure S5). In addition, when we examined mono- and polyubiquitinated proteins using the FK2 antibody, inhibition of the proteasome with MG132 caused the accumulation of high molecular weight proteins as anticipated. However, there was no difference seen in the quantity or mobility of EsxH_Mt (Figure S5). Further, when we mapped the region of Hrs required for the interaction with EsxG_Mt-EsxH_Mt in the Y2H assay, the UIM domain was not required. Amino acids 398–630, which contain a coiled-coil region, were sufficient to mediate the interaction (Figure 2E). We verified that the C-terminal half of Hrs was sufficient to mediate an interaction by co-immunoprecipitation (Fig. 3D). In summary, these data show that EsxG_Mt stabilizes EsxH_Mt in the mammalian cytosol and that the heterodimer can bind the C-terminus of Hrs.

To test the hypothesis that the EsxH_Mt interaction with Hrs disrupts ESCRT, we examined the effect of EsxG_Mt EsxH_Mt on epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) degradation (all performed without MG132 treatment). Upon binding ligand, EGFR is internalized and transferred into ILVs by ESCRT so that it can be degraded upon MVB-lysosome fusion. To determine whether EsxH_Mt interferes with this process, we transfected EsxG_Mt and EsxH_Mt or vector control into HEK293 cells and examined EGFR levels 90 min after EGF treatment. We found that EGFR levels decreased in control cells. In cells co-expressing EsxG_Mt and EsxH_Mt there was a 63+/−8% (n = 3) increase in the fraction of EGFR that remained undegraded (Figure 3E), similar to what has been seen with Hrs depletion [46]. In contrast, co-expression of EsxG_Ms and EsxH_Ms had no detectable effect on EGFR degradation (Figure 3E). We observed similar results when we used fluorescent EGF to examine trafficking in A549 cells using fluorescence microscopy. As expected, cells depleted of Hrs showed enhanced EGF fluorescence due to impaired degradation. We observed a similar decreased degradation in cells expressing EsxG_Mt and EsxH_Mt (Figure 3F and G). In contrast, expression of EsxG_Ms EsxH_Ms or EsxG_Mt EsxH_Mt-H76A-E77A had little effect on EGF degradation (Figure 3G and 3H). Thus, EsxH_Mt, in complex with EsxG_Mt, is sufficient to inhibit EGF and EGFR degradation, an activity that correlates with its binding to Hrs. EsxH_Ms, from the non-pathogenic species, does not have this property.

EsxG_Mt and EsxH_Mt arrest phagosome maturation

Because Esx-3 is essential for Mtb growth, we examined the effect of overexpressing EsxH_Mt on bacterial trafficking. First, we wanted to determine whether EsxG_Mt EsxH_Mt could confer a block in maturation to phagosomes containing Msmeg. However, when we expressed EsxG_Mt EsxH_Mt--FLAG under control of the hsp60 promoter in Msmeg, it was not secreted (Figure S6). It was secreted by Mtb (Figure 4A), and when we examined whether overexpression of EsxG_Mt and EsxH_Mt-FLAG could enhance phagosome maturation arrest of Mtb, we found less co-localization between Mtb and LAMP1 and enhanced co-localization with TfR with the strain overexpressing EsxH_Mt-FLAG, compared to a strain transformed with vector control (Figure 4B–E). The defect in lysosomal trafficking was similar to siRNA-mediated silencing of Hrs, Tsg101, and Rab7, and the combination of EsxG_Mt EsxH_Mt overexpression and ESCRT-silencing resulted in lower LAMP1 co-localization than either manipulation alone (Figure 4B). An Mtb strain that expressed EsxG_Mt EsxH_Mt- H76A-E77A did not exhibit altered trafficking, but the mutant protein also failed to be secreted (Figure 4A, 4F). Mtb did secrete EsxG_Ms EsxH_Ms-FLAG, which, unlike EsxG_Mt EsxH_Mt-FLAG, did not block LAMP1 co-localization (Figure 4A and 4F). We conclude that EsxG_Mt EsxH_Mt, but not EsxG_Ms EsxH_Ms, can prevent lysosomal trafficking during infection, most likely reflecting the ability of EsxH_Mt to bind Hrs and impair ESCRT activity.

Tissue culture conditions

RAW264.7 and HEK293 cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco), 20 mM HEPES, 2 mM L-glutamine, and 10% heat inactivated fetal bovine serum (hiFBS; Invitrogen). BMDMs were isolated from C57BL/6 mice as described [63] Penicillin/Streptomycin (Gibco), added for passaging, was omitted during infections. A549 cells were grown in RPMI 1640 Medium (Gibco), 2 mM L-glutamine, 1× Non-essential Amino Acids (Cellgro), and 10% hiFBS. Cells were grown at 37°C with 5% CO₂ atmosphere. siRNAs were transfected with Hiperfect (Qiagen). Plasmids were transfected into HEK293 cells with Effectene (Qiagen) and A549 cells with Lipofectamine 2000 (Invitrogen).

Bacterial strains and growth conditions

M. tuberculosis H37Rv, M. bovis-BCG, and M. smegmatis mc²155 were grown at 37°C to log phase in Middlebrook 7H9 media with 0.05% Tween 80, BBL Middlebrook OADC Enrichment, and 0.2% glycerol. Plasmids were selected with 50 µg/ml kanamycin or hygromycin depending upon the resistance marker. To generate EsxG_Mt EsxH_Mt-FLAG and EsxG_Ms EsxH_Ms-FLAG for overexpression in mycobacteria, EsxG-EsxH was amplified from BCG (the EsxG-EsxH region is 100% identical between BCG and Mtb) and Msmeg genomic DNA, respectively, using primers described in Text S1. The PCR products were cloned into pSYMP under control of the hsp60 promoter [64].

Intracellular bacterial growth assay

RAW cells were seeded one day before infection or they were transfected with siRNAs two days prior to infection with a single cell suspension of Mtb (MOI∼2–5), obtained as previously described [40]. The cells were extensively washed and lysed with 0.2% Triton X-100 3 hpi or 2d later and serial dilutions were plated on 7H10 or 7H11. CFU were calculated 15 to 21 d later.

Lysosomal trafficking assay

RAW cells were transfected with siRNAs for two days and then infected with a single cell suspension of BCG or Mtb (MOI∼20) for 3 h, then washed extensively. Cells were fixed 24 hpi with 4% formaldehyde/PBS for BCG and with 1% paraformaldehyde/PBS overnight for Mtb and immunostained for LAMP1 (Abcam) or TfR (Invitrogen). For Lysotracker (Invitrogen) staining, unfixed RAW cells were incubated with 200 nM Lysotracker, washed twice in PBS, and visualized. Images were captured using the Nikon Eclipse TiE/B automated fluorescent microscope with Photometrics HQ@ Monochrome digital camera. 60× z-stack images were acquired, deconvoluted, and analyzed using NIS-Elements DUO software (see Fig. S3 for details). Contrast was not altered prior to automated image analysis; for reproduced images, alterations were applied equally to all samples.

Recombinant protein binding assay

His-tagged EsxG_Mt-EsxH_Mt was purified as described in Text S1. Prior to inclusion of recombinant proteins in binding reactions they were centrifuged at 100,000× g for 30 min to remove aggregated protein. To determine whether EsxG_Mt EsxH_Mt binds to Hrs in a direct and saturable manner, 1.0 µg EsxG_Mt EsxH_Mt--6XHis was bound to Ni-NTA beads and incubated with increasing amounts of purified, soluble Hrs (0∼4 µg) in 20 mM HEPES [pH, 7.4], 150 mM KCl, and 0.05% Tween-20, with protease inhibitors (10 mM leupeptin, 1 µg/µL pepstatin, 0.3 mM aprotinin, and 1.74 µg/µL PMSF) for 1 h at 4°C. Beads were washed in PBST (0.1 M PBS 0.05% Tween-20) with 10 mM imidazole. Bound Hrs was analyzed by SDS-PAGE and Coomassie staining. Bands were subject to quantification with ImageJ software (v. 1.42).

Co-immunoprecipitation and Western blotting

Cellular lysates were prepared in RIPA buffer with Halt Protease Inhibitor Cocktail (Thermo Scientific) and 10 mM N-ethylmaleimide (Sigma) and analyzed by western blotting. The antibodies used for western analysis are: actin (clone C4/MAB1501, Millipore), Hrs (M79/sc-30221, Santa Cruz Biotechnology), Rab7 (117, Abcam), FLAG (F7425, Sigma), EGFR (#4267S, Cell Signaling), and FK2 (Millipore). For co-immunoprecipitation, HEK293 cells transfected with Hrs-myc and Esx expression plasmids were treated with 20 µM MG132 (Calbiochem) for 3 h prior to mechanical lysis and incubated with Dynabeads Protein G (Novex, Life technologies) pre-bound to isotype control antibody (sc-2025, Santa Cruz Biotechnology), anti-myc antibody (sc-40/9E10, Santa Cruz Biotechnology), or anti-V5 antibody (Invitrogen), and bound proteins were analyzed by western blotting.

EGFR and EGF degradation assays

Two days after transfection, HEK293 cells were incubated in serum-free DMEM and treated with 100 ng/ml of recombinant human EGF (rh-EGF, R&D Systems) essentially as described [65]. Cells were harvested immediately prior to addition of EGF and 90 min later and EGFR analyzed by western blotting. EGF trafficking was assessed similarly to described [66]. Two days after transfection of A549 cells with siRNA or DNA, cells were incubated with serum-free RPMI before addition of 25 µg/ml Alexa Fluor 488-EGF (Invitrogen) in EGF uptake media (RPMI, 2% BSA, 20 mM HEPES) at 4°C for 1 h. Cells were washed to remove unbound ligand, incubated at 37°C for 90 min, and examined by immunofluorescence microscopy.

Mycobacterial secretion of EsxH

To analyze secretion of EsxH_Ms-FLAG or EsxH_Mt-FLAG from Mtb and Msmeg, strains were grown to mid-log phase, washed with PBS, and inoculated into Sauton's media. In Sauton's media, they were grown to reach log phase (overnight in the case of Msmeg and for two days in case of Mtb). Thereafter, mycobacteria were pelleted by centrifugation. The supernatants were filtered through 0.22 µM filters followed by precipitation with 12% trichloroacetic acid. The precipitate was washed with ice-cold acetone, air dried, and resuspended in SDS sample buffer. The bacterial pellets were lysed by bead beating in lysis buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 0.6% SDS, 10 mM NaH₂PO₄, and protease inhibitor) with 0.1 mm zirconia/silica beads (BiosSpec Products, Inc.). SDS-sample buffer was added, followed by boiling at 95°C for 5 min. Antibody to the pyruvate dehydrogenase E2 component sucB (Rv2215/dlaT) [67], a cytosolic protein, was used as a loading control and to indicate the degree of bacterial lysis.