Mtb encodes several protein secretion systems important for bacterial virulence (Feltcher et al., 2010). One of the type VII secretion systems (T7SS) of Mtb, contained within the region of difference 1 (RD1) locus of Mtb (Behr et al., 1999), secretes virulence factors including EsxA and EsxB (also known as CFP-10) (Stanley et al., 2003; Figure 1A). We hypothesized that the Mtb T7SS might facilitate M cell translocation because the T7SS machine interacts directly with eukaryotic membranes (Augenstreich et al., 2017), EsxA can be identified on the mycobacterial cell surface (Kinhikar et al., 2010), and EsxA may directly bind several cell surface receptors (Kinhikar et al., 2010; Sreejit et al., 2014). To test if the Mtb T7SS was required for bacterial binding to and translocation across M cells, we used a human airway M cell transwell model that we developed previously (Figure 1B; Nair et al., 2016). Such transwells combine 16-HBE cells (Cozens et al., 1994) in the apical compartment of a transwell and Raji B cells in the basolateral compartment in order to mimic the organization of MALT and to enhance M cell formation (Kernéis et al., 1997; Nair et al., 2016). HBE cells cultured alone form a homogenous, polarized monolayer (hereafter called ‘control’) while coculture with Raji B cells induces some HBE cells to differentiate into M cells (hereafter called ‘HBE/Raji B’) (Nair et al., 2016). We used the Mtb eccD1::Tn5370 strain (hereafter designated Mtb eccD1::Tn) (Cox et al., 1999; Stanley et al., 2003), which lacks the inner membrane pore required for assembly of and protein secretion by the T7SS (Figure 1A; Abdallah et al., 2007). To test if the Mtb T7SS was necessary for M cell binding, we incubated transwells with either wild-type Mtb expressing mCherry (WT Mtb) or Mtb eccD1::Tn expressing mCherry at 4°C to prevent bacterial entry or translocation and analyzed surface binding by confocal microscopy and quantification of colony-forming units (CFU) (Figure 1C–E). Consistent with our prior data (Nair et al., 2016), significantly more WT Mtb bound HBE/Raji B transwells (containing M cells) than control transwells by both microscopy (Figure 1C,D) and CFU (Figure 1E). However, binding was greatly reduced for the Mtb eccD1::Tn strain (Figure 1C–E). To confirm that the Mtb eccD1::Tn strain did not harbor additional mutations other than the expected transposon insertion into eccD1 to potentially explain this result, we performed whole genome sequencing on the Mtb Erdman WT lab strain from our lab, along with the Mtb eccD1::Tn strain. No significant insertions, deletions or mutations were observed in the Mtb eccD1::Tn strain relative to the wild-type strain, although several single-nucleotide polymorphisms were noted when we compared the strains to the reference strain (Miyoshi-Akiyama et al., 2012; Supplementary file 1).

To test if the Mtb T7SS is necessary to facilitate mycobacterial translocation across M cells, we infected the apical chamber of transwells with either WT Mtb or Mtb eccD1::Tn and measured translocation to the basal compartment. As we reported previously (Nair et al., 2016), WT Mtb translocated across HBE/Raji B transwells to a greater extent than control transwells, while the translocation of the Mtb eccD1::Tn strain was significantly reduced (Figure 1F). Importantly, the transepithelial electrical resistance (TEER), a measure of epithelial monolayer integrity (Srinivasan et al., 2015), was stable during the experiment (Figure 1G). To further verify that Mtb T7SS is required for bacterial translocation across M cells in vitro, we also utilized an established model of M cell differentiation where Caco-2 cells, a human colonic epithelial cell line, are cultured with Raji B transwells to induce M cell differentiation (Nair et al., 2016). Similar to HBE/Raji B transwells, we observed that Mtb translocated across Caco-2/Raji B transwells in a T7SS-dependent manner (Figure 1H,I). Taken together, these data show that the Mtb T7SS is necessary for both binding to and translocation across M cells in vitro.

Two of the most abundant T7SS secreted proteins are EsxA and EsxB (Berthet et al., 1998; Brodin et al., 2005; Champion et al., 2014); therefore, we hypothesized that one of these proteins might mediate Mtb binding and translocation. We expressed EsxA and EsxB as 6-His tagged constructs and purified the proteins from E. coli (Figure 2—figure supplement 1). We then conjugated recombinant EsxA, EsxB, or glycine (as a control) to fluorescent beads, added the beads to the apical chamber of transwells, and quantified bead translocation to the basal compartment by flow cytometry. EsxA-beads but not control beads translocated across HBE/Raji B transwells (Figure 2A) without disrupting the epithelial monolayer (Figure 2B). EsxA-beads, but not EsxB-beads or control beads, also translocated across Caco-2/Raji B transwells (Figure 2C).

To test if the ability of EsxA to mediate translocation was due to direct EsxA binding to M cells, we incubated transwells with recombinant 6xHis-tagged EsxA or EsxB and performed immunofluorescence microscopy using antibodies against 6x-His and α1,2-fucose (NKM 16-2-4; a marker for M cells [Nair et al., 2016; Nochi et al., 2007; Figure 2D]). While both groups of transwells had equal number of nuclei per field (Figure 2E), HBE/Raji B transwells had more NKM 16-2-4-positive M cells compared to control transwells (Figure 2F). We detected robust EsxA binding to M cells on HBE/Raji B transwells, in contrast to EsxB, which did not demonstrate significant binding (Figure 2D,G–H). Similar results were observed using an antibody against Sialyl Lewis^A (SLA), a different M cell marker (Giannasca et al., 1999; Nair et al., 2016; Figure 2—figure supplement 2). Taken together, these data demonstrate that EsxA, but not EsxB, directly binds the M cell surface and is sufficient to mediate translocation across M cells when conjugated to inert beads.

Because EsxA bound directly to the surface of M cells, we hypothesized that EsxA may engage a cell surface receptor. To affinity purify cell surface binding proteins, we performed a modified co-immunoprecipitation experiment using either EsxA or transferrin crosslinked to the TriCEPS reagent, a molecule that allows for the covalent cross-linking of a ligand and its receptor (Tremblay and Hill, 2017). We used Caco-2 cells for this experiment as they have been used extensively as a model for M cells in vitro (Tyrer et al., 2006) and because Caco-2/Raji B transwells behaved similarly to HBE/Raji B transwells in Mtb and EsxA translocation (Figure 1E,G and Figure 2A,C). Using this approach, we identified the interaction between transferrin (TRFE) and the transferrin receptor (TFR1) (Figure 3A, blue peptides; Supplementary file 2), proving the validity of this system. When cells were treated with EsxA, peptides for two proteins, apolipoprotein E (ApoE) and scavenger receptor class B type I (SR-B1) were enriched (Figure 3A, red peptides; Supplementary file 2). Because ApoE is a soluble protein (Huang and Mahley, 2014) while SR-B1 is a known cell surface molecule, we focused on SR-B1. For further verification, we performed a co-immunoprecipitation/biotin transfer experiment without the TriCEPS reagent. After incubation with biotinylated EsxA, completion of the biotin transfer assay, and subsequent immunoprecipitation with streptavidin-coated beads, western blotting with an anti-SR-B1 antibody detected an approximately 82 kD band consistent with the known observed molecular weight of glycosylated SR-B1 (Acton et al., 1996; Figure 3B).

We next determined if SR-B1 expression is specific for M cells or ubiquitously expressed by epithelial cells. We quantified colocalization of SR-B1 and NKM 16-2-4 by immunofluorescence microscopy in control and HBE/Raji B transwells (Figure 3C–F). While there was no difference in the number of nuclei per field on the transwells (Figure 3C), SR-B1 expression was higher on HBE/Raji B transwells (Figure 3C,E) and the majority of the SR-B1 positive cells were NKM⁺ M cells (Figure 3C,F). Taken together, we identify SR-B1 as a candidate EsxA receptor expressed on M cells in vitro.

We next investigated whether SR-B1 is required for EsxA binding to M cells by exposing cells to recombinant EsxA in the presence or absence of SR-B1. We first transduced HBE cells with non-targeting (NT) or SR-B1 shRNA and observed a robust knock-down of SR-B1 in HBE cells transduced with the SR-B1 shRNA as compared to the NT shRNA (Figure 4A). HBE/Raji B transwells constructed from these cells had a similar number of M cells comparing NT and SR-B1 shRNA transwells (Figure 4B,C) and SR-B1 shRNA transwells showed a reduction in SR-B1 expression by immunofluorescence microscopy (Figure 4B,D). When we incubated these transwells with recombinant EsxA, absence of SR-B1 reduced the number of EsxA positive cells on HBE/Raji B SR-B1 shRNA transwells (Figure 4B,E). Additionally in HBE/RajiB NT shRNA transwells, the majority of EsxA-positive cells were SR-B1 positive (Figure 4B,F), suggesting that EsxA preferentially bound SR-B1 expressing M cells. Taken together, we identify SR-B1 as necessary for EsxA binding to M cells.

To determine the role of SR-B1 in Mtb binding to M cells, we incubated HBE/Raji B transwells expressing NT or SR-B1 shRNAs with mCherry Mtb at 4°C for 1 hr and analyzed binding by confocal microscopy and CFU (Figure 4G–I). Loss of SR-B1 greatly reduced the number of bacteria bound to the HBE/Raji B transwells as determined by confocal microscopy (Figure 4G,H) and by quantification of CFU (Figure 4I). To determine the role of SR-B1 in Mtb translocation by M cells, we infected HBE/Raji B transwells expressing NT or SR-B1 shRNAs with Mtb in the apical compartment and measured translocation to the basal compartment. As expected from the reduced bacterial binding (Figure 4G–I), loss of SR-B1 also greatly reduced Mtb translocation in the HBE/Raji B transwells at 37°C (Figure 4J) with no impact on the TEER (Figure 4K). The reduced ability of Mtb to translocate across HBE/Raji B transwells expressing SR-B1 shRNA was not due to any intrinsic defect in translocation caused by SR-B1 deficiency as another airway pathogen, Pseudomonas aeruginosa, was able to translocate equally across NT and SR-B1 shRNA HBE/Raji B transwells (Figure 4L). Of note, P. aeruginosa does not encode a T7SS or EsxA homologue, suggesting that its translocation across M cells depends on unique bacterial and host factors. We thus conclude that SR-B1 is essential for the binding and translocation of Mtb via M cells in a process requiring the effector EsxA.

M cells are found in the upper and lower airways of mice and humans (Fujimura, 2000; Mutoh et al., 2016; Nair et al., 2016; Teitelbaum et al., 1999). We therefore determined if SR-B1 was expressed preferentially by primary M cells as compared to other epithelial cells using immunofluorescence microscopy. Mouse nasal-associated lymphoid tissue (NALT), a region enriched for M cells (Mutoh et al., 2016; Nair et al., 2016; Park et al., 2003), demonstrated robust SR-B1 staining on the surface of NKM 16-2-4 positive cells (Figure 5A). Importantly, we did not observe SR-B1⁺/NKM 16-2-4^- cells, demonstrating that SR-B1 is specific for M cells in the NALT epithelia in vivo.

We and others previously demonstrated that NALT and airway M cells are a portal of entry for Mtb in mice (Nair et al., 2016; Teitelbaum et al., 1999). To determine if the Mtb T7SS is necessary for bacterial translocation in vivo, we performed NALT infections (Nair et al., 2016) with Mtb eccD1::Tn or MtbΔesxA. As described above for Mtb eccD1::Tn, to control for possible mutations outside the known esxA deletion, we performed whole genome sequencing on the Mtb∆esxA strain and determined that its sequence is essentially identical to the parental Mtb WT strain (Supplementary file 1). Of note, in both Mtb mutant strains the T7SS machine fails to assemble (Abdallah et al., 2007) thereby preventing T7SS-dependent virulence factor secretion. We infected mice intranasally with WT Mtb, Mtb eccD1::Tn (Figure 5B), or MtbΔesxA (Figure 5C) and enumerated CFU from draining cervical lymph nodes 7 days post-infection (Nair et al., 2016). Both the Mtb eccD1::Tn and Mtb∆esxA strains had 1.0–1.5 log fewer bacteria compared to WT Mtb in the cervical lymph nodes. This degree of attenuation was not observed when we infected mice intranasally with a Cor-deficient strain of Mtb (Mtbcor::Tn7) that is also attenuated in vivo (Zacharia et al., 2013; Figure 5—figure supplement 1). Taken together, these data demonstrate that the lower CFU recovered from cervical lymph nodes of mice infected with T7SS mutant Mtb strains may be due to reduced translocation across M cells.

Because TB is a human disease, we tested if primary human M cells can serve as a portal of entry for Mtb. The human adenoid is a MALT structure that contains M cells interspersed among the overlying epithelial cells (Fujimura, 2000). We first demonstrated that human adenoids contain SR-B1⁺/NKM⁺ M cells by immunofluorescence microscopy (Figure 5D), similar to our observations from mouse NALT (Figure 5A). We next confirmed the presence of M cells in human adenoids by flow cytometry (Figure 5—figure supplement 2 for gating strategy) and observed that approximately 10% of adenoid cells were EpCAM⁺/NKM^- epithelial cells, while about 1% of the cells were EpCAM⁺/NKM⁺ double positive M cells (Figure 5E). When we analyzed SR-B1 expression using flow cytometry, we observed that approximately 20% of primary human M cells (marked as EpCAM⁺/NKM⁺ cells) were SR-B1 positive as compared to less than 2% of the other epithelial cells (EpCAM⁺/NKM^-) (Figure 5F), verifying our observation that SR-B1 is expressed predominately by M cells in vivo.

To determine if primary human M cells can be a route of entry for Mtb, we infected human adenoids with GFP⁺ Mtb and quantified the number of GFP⁺ Mtb EpCAM⁺/NKM⁺ M cells versus GFP⁺ Mtb EpCAM⁺/NKM^- epithelial cells by flow cytometry (Figure 5G, Figure 5—figure supplement 3 for gating strategy). The number of GFP⁺ Mtb containing EpCAM⁺/NKM⁺ M cells ranged from 0.1–8% while we were unable to identify any GFP⁺ Mtb containing EpCAM⁺/NKM^- epithelial cells (Figure 5G), suggesting that M cells were a preferred route of entry for Mtb in adenoids. Finally, we determined the role of the Mtb T7SS in Mtb entry into adenoid M cells. We infected adenoids with GFP⁺ WT Mtb or GFP⁺ Mtb eccD1::Tn and observed more GFP⁺/EpCAM⁺/NKM⁺ cells after infection with WT Mtb as compared to Mtb eccD1::Tn (Figure 5H). Taken together, we conclude that Mtb can enter via mouse NALT and human adenoid M cells in a T7SS dependent manner.

M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370 (Cox et al., 1999; Stanley et al., 2003), M. tuberculosis Erdman ΔesxA (Stanley et al., 2003), M. tuberculosis Erdman cor:Tn7 (Zacharia et al., 2013) were grown in Middlebrook 7H9 medium or on Middlebrook 7H11 plates supplemented with 10% oleic acid-albumin-dextrose-catalase. Tween 80 (Fisher T164-500) was added to liquid medium to a final concentration of 0.05%. Strains M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370 and M. tuberculosis Erdman ΔesxA underwent whole genome sequencing to determine the presence of unknown genetic polymorphisms.

M. tuberculosis Erdman, M. tuberculosis Erdman eccD1::Tn5370, M. tuberculosis Erdman ΔesxA were grown to late-log phase and genomic DNA isolated by the cetyltrimethylammonium bromide (CTAB)-lysozyme method (Larsen et al., 2007). Genomic DNA was then enzymatically fragmented and sequenced using Illumina NextSeq 550 sequencing (Microbial Genome Sequencing Center, https://migscenter.com). To reconstruct the bacterial genomes and identify genetic modifications, the bioinformatics analysis workflow was based on Genome Analysis Toolkit (GATK, v3.8–0; RRID:SCR_001876) (DePristo et al., 2011; McKenna et al., 2010) best practices. Quality control and adapter trimming were performed using Trim Galore (v0.6.4; RRID:SCR_011847) (https://github.com/FelixKrueger/TrimGalore). Burrows-Wheeler Aligner (BWA, v0.7.17; RRID:SCR_010910) (Li and Durbin, 2009) was employed to map the reads to the genome of the publicly available Mtb Erdman (ATCC35801) strain (NCBI assembly: ASM35020v1). Picard (v2.12.0; RRID:SCR_006525) (https://broadinstitute.github.io/picard) was used to remove PCR indices. Variant calling and genotyping were performed using GATK HaplotypeCaller (RRID:SCR_001876) and the variant calls were filtered by applying the following criteria: DP (Approximate read depth)<10, GQ (Genotype Quality)<20. The variants were annotated using a custom Perl script (https://github.com/jiwoongbio/Annomen). Insertions and deletions were identified using coverage depths and split reads from SAMtools (v0.1.19; RRID:SCR_002105) (Li et al., 2009). SPAdes (v3.13.0; RRID:SCR_000131) (Bankevich et al., 2012) was used to de novo assembly and MUMmer 4 (RRID:SCR_001200) (Kurtz et al., 2004) was used to compare the genome assemblies. The genomes are available at NCBI Sequence Read Archive Accession #PRJNA605439.

The human colorectal adenocarcinoma cell line Caco-2 (HTB-37; RRID:CVCL_0025) and human Burkitt lymphoma cell line Raji B (CCL-86; RRID:CVCL_0511) were obtained from ATCC (Manassas, VA). 16HBE14o- cells (RRID:CVCL_0112) (Forbes et al., 2003) were provided by Dieter Gruenert (University of California, San Francisco). Caco-2 or HBE cells were grown in DMEM (Gibco 11965092) supplemented with 20% fetal bovine serum (Gibco 26140079), 50 units/mL penicillin (Gibco 15140122), 50 μg/mL streptomycin (Gibco 15140122), 2 mM L-glutamine (Gibco 25030081), 1% sodium pyruvate (Gibco 11360070), 1% non-essential amino acids (Gibco 11140050), and 1 mM HEPES (Hyclone SH30237.01). Raji B cells were grown in DMEM supplemented with 20% FBS and 2 mM L-glutamine. In order to generate stable knock-down lines of SR-B1, HBE cells were transduced with lentivirus containing the appropriate shRNA cloned into pLKO.1 (Addgene 10878) as described previously (Huang et al., 2019). Transduced cells were selected with puromycin (Sigma-Aldrich P8833-10MG) and surviving cells were maintained in puromycin for three additional passages. All cell lines were routinely tested for mycoplasma contamination. HBE, Caco-2 and Raji B cell lines were verified using STR profiling analysis (ATCC).

3 × 10⁵ Caco-2 or HBE cells in 1 mL of media were plated in the upper chamber of a 3 μm transwell insert (Corning 3462). For Raji B treated transwells, 5 × 10⁵ Raji B cells in 2 mL of media were added to the basal compartment, thereby inducing some of the overlying epithelial cells to differentiate into M cells. For control transwells, 2 mL of media alone were added to the basal chamber, leading to little to no M cell differentiation. 1 mL of media in the upper chamber and 1 mL of media in the bottom chamber were aspirated daily and replaced with 1 mL of fresh media. The transwells were maintained at 37°C for 2 weeks or until the transepithelial electrical resistance was greater than 350 Ω. 72 hr prior to infection, transwells were cultured in media lacking antibiotics. Transwell media was changed approximately 2 hr prior to infection.

Liquid cultures of Mtb were grown until mid-log phase, washed three times with PBS, and centrifuged and sonicated to remove clumps. Bacteria were then resuspended in DMEM + 20% fetal bovine serum. For translocation assays, bacterial inoculum was added to the upper chamber of the transwell at a MOI of 5:1 and media from the basal compartment was sampled after 60 min. The samples were then plated on 7H11 agar plates and maintained in a 37°C incubator for 3 weeks to allow for colony formation.

gBlocks (IDT) encoding Mtb EsxA or EsxB were first cloned into the pENTR entry vector (Thermo K240020) then subcloned into the pDest17 destination vector (Thermo 11803012; Thermo 11791020) using Gateway cloning (Invitrogen) per the manufacturer’s protocol. The resulting vectors were cloned into the BL21 strain of E. coli (NEB C2527I) for protein expression. 1 L of bacterial culture was grown to an OD600 of 0.6, induced with 1 mM IPTG (Promega V3955) at 37°C for 3 hr, and centrifuged at 3500 rpm for 15 min at 4°C to yield a bacterial pellet. The bacterial pellet was then resuspended in 15 mL of resuspension buffer (50 mM sodium phosphate, 500 mM NaCl, pH 7.4) with one tablet of EDTA-free protease inhibitor (Roche 11836170001). Bacteria were lysed by sonication and centrifuged at 11,200 rpm for 15 min at 4°C. The resulting pellet was resuspended in 20 mL of 8 M urea in resuspension buffer and incubated for 2 hr at room temperature with gentle agitation. The protein slurry was again centrifuged at 11200 rpm for 15 min at 4°C and the resulting supernatant was incubated with cobalt TALON affinity resin (Clontech 635503) for 2 hr at room temperature. Resin was washed with 8 M urea in resuspension buffer and EsxA or EsxB was eluted with 150 mM imidazole and 8 M urea in resuspension buffer. The eluate was dialyzed overnight using a Slide-a-Lyzer dialysis cassette (Thermo 66203) against 10 mM ammonium bicarbonate. The dialyzed sample was again incubated with cobalt TALON affinity resin for 2 hr at room temperature. Resin was subsequently washed with 10 mM Tris-HCl pH 8.0, 0.5% ASB-14 (Sigma A1346-1G) in 10 mM Tris-HCl pH 8.0, and 10 mM Tris-HCl pH 8.0. EsxA or EsxB was eluted with 150 mM imidazole in PBS, dialyzed overnight against PBS, and stored at 4°C. Fractions were analyzed by SDS-PAGE followed by Coommassie staining with Brilliant Blue R-250 (Fisher BP101-25).

In order to image binding of Mtb to transwells, mCherry Mtb was grown until mid-log phase, washed, and centrifuged and sonicated to remove clumps. The bacterial inoculum was added to the upper chamber of the transwell at a MOI of 5:1 for 2 hr at 4°C with gentle agitation every 15 min. Transwells were gently washed and fixed with 4% paraformaldehyde in PBS at 4°C for 1 hr. Transwell inserts were stained with DAPI (Thermo D1306), excised using a blade, mounted on microscope slides using Prolong Gold antifade reagent (Invitrogen P36390) and imaged using an AxioImager MN microscope (Zeiss). In order to image binding of EsxA to transwells, EsxA was expressed and purified as described above. EsxA was then biotinylated by the Sulfo-SBED reagent (Thermo 33033) per manufacturer’s instructions and excess reagent was removed using PD-10 desalting columns (GE Healthcare 17-0851-01). Transwells were then incubated with 1.5 μM EsxA in HBSS for 2 hr at 4°C with gentle agitation, washed, and exposed to UV light for 30 min at room temperature to allow for cross-linking. Transwells were then fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, blocked with 10% donkey serum (Sigma D9663-10ML) in PBS for three hours at room temperature, and incubated with a 1:100 dilution of rabbit anti-SR-B1 antibody (Abcam 52629; RRID:AB_882458) in 2% donkey serum in PBS overnight at 4°C. The following day, transwells were washed and incubated with a 1:100 dilution of PE-conjugated rat NKM 16-2-4 (Miltenyi 130-102-150; RRID:AB_2660295), a 1:500 dilution of an AlexaFluor 647 conjugated donkey-anti-rabbit secondary antibody (Thermo A-31573; RRID:AB_2536183), and a 1:500 dilution of AlexaFluor 488 conjugated streptavidin (Jackson 016-540-084; RRID:AB_2337249) for 1 hr at room temperature. Transwells were then washed, stained with DAPI, excised with a blade, mounted, and imaged as described above. Five fields of view were imaged per independent experiment.

1 µm microspheres were conjugated to protein as per instructions (Polylink 24350–1). Briefly, 12.5 mg of microspheres were centrifuged and washed twice in coupling buffer. Microspheres were then incubated with an EDAC/coupling buffer solution to activate the microspheres. 200 μg of protein is added to the beads, thereby allowing for covalent binding of the protein to the microspheres. Microspheres are then washed twice with PBS and stored at 4°C. In order to test the ability of these beads to translocate in the tissue bilayer assay, beads were diluted to a MOI of 5:1 in DMEM + 20% fetal bovine serum and added to the apical chamber of transwells. Media from the basal compartment was sampled after 60 min and the number of beads present in the sample was analyzed by flow cytometry using an LSR II flow cytometer (BD).

For initial conjugation of TriCEPS to protein, EsxA or transferrin (300 μg) dissolved in 150 μL 25 mM HEPES pH 8.2 buffer was added to 1.5 μL of the TriCEPS reagent (Dualsystems Biotech) and incubated at 20°C for 90 min with gentle agitation. During this time, 6 × 10⁸ Caco-2 cells were detached from tissue culture plates using 10 mM EDTA in PBS. Cells were split into three aliquots, cooled to 4°C, and pelleted. Each pellet was resuspended in PBS pH 6.5 and sodium metaperiodate was added to a final concentration of 1.5 mM in order to gently oxidize the cell surface. Cells were then incubated with sodium metaperiodate in the dark for 15 min at 4°C. Cells were washed twice with PBS pH 6.5 and split into two new aliquots. TriCEPS coupled EsxA was added to one aliquot and TriCEPS coupled transferrin was added to the other aliquot and incubated for 90 min at 4°C with gentle agitation. Samples were then washed, lysed via sonication, and digested with trypsin. The TriCEPS reagent:ligand:receptor complex was then affinity purified and samples were analyzed using a Thermo LTQ Orbitrap XL spectrometer fitted with an electrospray ion source. Samples were measured in data-dependent acquisition mode in a 90 min gradient using a 10 cm C18 packed column. Samples were analyzed with a statistical ANOVA model with p-values adjusted to control the experiment-wide false discovery rate (FDR). The adjusted p-value obtained for each protein was plotted against the fold enrichment between the two experimental conditions. The area in the volcano plot with an enrichment factor of 4 fold or greater and an FDR-adjusted p-value less than or equal to 0.01 was defined as the receptor candidate space.

EsxA was expressed and purified as described above. EsxA or PBS alone was then biotinylated by the Sulfo-SBED reagent (Thermo 33033) according to the manufacturer instructions and excess reagent was removed using PD-10 columns (GE Healthcare 17-0851-01). 1 × 10⁷ HBE cells were detached from tissue culture plates using 10 mM EDTA in PBS. Cells were washed, resuspended in HBSS, and incubated with 1.5 μM EsxA or with PBS alone for 2 hr at 4°C with gentle agitation. Cells were then washed and exposed to UV light for 30 min at room temperature to allow for covalent cross-linking. Cells were lysed with RIPA buffer and lysate was incubated with streptavidin-conjugated magnetic beads (Thermo 88816). Proteins were eluted by boiling and analyzed by SDS-PAGE followed by western blotting with rabbit anti-SR-B1 antibody (Abcam 52629; RRID:AB_882458).

Mouse NALT sections were obtained as previously described (Nair et al., 2016). Briefly, mouse NALT (after decalcification) and human adenoid specimens were embedded in paraffin, sectioned (5 μm), and mounted on glass slides. Slides were deparaffinized using xylene and ethanol washes followed by heat mediated antigen-retrieval in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was quenched and slides were blocked in 10% donkey serum in PBS for 3 hr at room temperature. Slides were washed with PBS and incubated with a 1:100 dilution of mouse NKM 16-2-4 and rabbit anti-SR-B1 in 2% donkey serum in PBS overnight at 4°C. Slides were then washed with PBS and incubated with a 1:500 dilution of AlexaFluor 568 conjugated goat-anti-mouse secondary antibody (Thermo A-11004; RRID:AB_2534072) or with HRP-conjugated donkey-anti-rabbit secondary antibody (Thermo A16023; RRID:AB_2534697) in 2% donkey serum in PBS for 1 hr at room temperature. Slides were then washed with PBS and incubated with Cy5 tyramide (Perkin Elmer SAT705A001EA) for 8 min. Slides were then washed with PBS, incubated with DAPI, washed with PBS, mounted in Prolong Gold antifade reagent, and imaged using an AxioImager MN microscope (Zeiss). At least three fields of view were imaged per independent experiment.

Mtb Erdman and all mutants were grown in 7H9 and 0.05% Tween-80 until mid-log phase. Cultures were washed three times with PBS, centrifuged to remove clumps, and sonicated to yield a single-cell suspension. Bacteria were resuspended to yield a final concentration of 1 × 10⁸ bacteria in 10 μL PBS. BALB/c mice obtained from The Jackson Laboratory (RRID:IMSR_JAX:000651) were infected with 10 μL of the bacterial suspension intranasally. NALT from three to five mice were collected, homogenized, and plated on 7H11 (Difco 283810) plates supplemented with 10% OADC to enumerate the number of bacteria deposited on Day 0. Mice were sacrificed on Day 7 post-infection and cervical lymph nodes were collected, homogenized, and plated on 7H11 plates. Plates were incubated in a 37°C incubator for 3 weeks to allow for colony formation.

Adenoid samples were obtained from children undergoing elective adenoidectomy for obstructive sleep apnea. Excised adenoids were immediately placed in DMEM, subsequently dissected into 3–4 pieces depending on the size of the adenoid, weighed, and mounted in a 2% agar pad such that only the mucosal surface was exposed. The adenoid pieces were then incubated overnight at 37°C in DMEM supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 1% sodium pyruvate, 1% non-essential amino acids, 1 mM HEPES, 50 ug/mL kanamycin, and 50 ug/mL ampicillin to kill commensal bacteria. The following morning, liquid cultures of GFP Mtb (Kanamycin-resistant) grown to mid-log phase were washed three times with PBS and centrifuged and sonicated to remove clumps. Bacteria were then diluted to 1 × 10⁷ bacteria/mL and 1 mL of inoculum was added to the adenoid and incubated at 37°C for 1 hr. Adenoids were then washed, minced into small pieces, and pushed through a 100 μm nylon cell strainer (Corning 431752). Cells were centrifuged, washed in ACK (Ammonium-Chloride-Potassium) lysis buffer (Gibco A10492-01), and then resuspended in FACS buffer (PBS + 2% FBS). Cells were stained with a 1:100 dilution of mouse anti-EpCAM Brilliant Violet 421 (Biolegend 324219; RRID:AB_11124342), mouse PE-NKM-16-2-4 (Miltenyi 130-102-150; RRID:AB_2660295), or rabbit anti-SR-B1 in FACS buffer, washed, and then incubated with a 1:500 dilution of AlexaFluor 488 conjugated donkey-anti-rabbit secondary antibody (Thermo R37118; RRID:AB_2556546). Cells were washed and fixed in 4% paraformaldehyde for 1 hr followed by counting on an LSRII flow cytometer and analyzed using FlowJo software.

Statistical analysis was performed using GraphPad Prism (RRID:SCR_002798). For in vitro transwell infections to determine bacterial binding or translocation, one-way ANOVA with corrections for multiple comparisons was performed. For in vitro determination of antibody staining, one-way ANOVA with corrections for multiple comparisons was performed. For in vivo adenoid infections or receptor expression, the paired non-parametric Wilcoxon matched pairs signed rank test was performed. For in vivo mouse infections and determination of CFU, the non-parametric Mann-Whitney U test was performed.