The M.tb H37Rv ΔmbtB::hyg, siderophore knockout strain was received as a kind gift from Dr. CE Barry, Tuberculosis Research Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Disease, Rockville, Maryland (De Voss et al., 2000). M.tb H37Ra and H37Rv strains were transformed with pSC301 plasmid (Cowley and Av-Gay, 2001) to generate the M.tb H37Ra-GFP and M.tb H37Rv-GFP strains. M.tb H37Ra strains expressing mCherry or GAPDH-mCherry were prepared previously in our laboratory (Boradia et al., 2014).

The full length M.tb GAPDH gene was cloned and expressed in M.tb H37Ra to obtain recombinant wild type GAPDH (wt rGAPDH) as described previously (Boradia et al., 2016). During plasmid screening two point mutations were detected (i) an Arginine to Serine mutation at position 142 (N142S) and (ii) a Proline to Leucine mutation at position 295 (P295L). Each plasmid was individually transformed into M.tb H37Ra using standard methods (Wards and Collins, 1996), recombinant proteins were purified as described previously (Boradia et al., 2016), the mutant proteins are referred to as rGAPDH(N142S) and rGAPDH(P295L) respectively.

Human Lf (Sigma Chemical Co.) was biotinylated using Sulfo-NHS-LC-Biotin as per the manufacturer's instructions (Pierce). Hilyte Fluor (HF)-488 was conjugated to Lf using AnaTag™ HiLyte Fluor™ 488 Protein Labeling Kit according to the manufacturer's instructions.

M.tb H37Ra cells were cultured to log phase, 2 × 10⁸ cells were used per assay. Cells were washed with PBS and blocked for 1 h at 4°C with PBS containing 2% BSA. Cells were then incubated with 10 μg Lf-HF488 alone or in the presence of 100-fold excess unlabeled human holo-Lf at 4°C for 2 h. Finally, cells were washed extensively with PBS and the fluorescence data of 10⁴ cells per assay was acquired using a Guava 8HT flow cytometer. Experiments were repeated thrice, statistical significance was determined by Student's t-test.

To assess Lf binding, M.tb H37Ra bacilli were cultured to log phase, 2 × 10⁸ cells were used per assay. Cells were washed with PBS and blocked in PBS containing 2% casein at 4°C for 1 h. Cells were then incubated with 10 μg of Lf- gold (20 nm) in PBS containing 0.2% casein at 4°C for 2 h. Cells were washed and fixed in Karnovsky's fixative for 30 min, followed by two washes with 5 mM NaCl. Finally, cells were resuspended in PBS, placed on carbon coated grids and viewed in a JEOL 2100 Transmission Electron Microscope (TEM). As a negative control, samples were similarly incubated with streptavidin gold conjugate (20 nm, Sigma) instead of Lf. Gold particles were prepared as described previously (Boradia et al., 2014).

M.tb H37Ra mCherry and M.tb H37Ra GAPDH mCherry expressing cells were cultured to log phase, 1 × 10⁸ cells were used per assay. Cells were washed with iron free Sauton's media and blocked for 1 h at 4°C with 2% BSA in PBS. Cells were then incubated with Lf conjugated to Alexa fluor 647 (Lf-A647, 10 μg per 100 μl of iron free Sauton's media) at 4°C for 2 h. Finally, cells were washed extensively with iron free Sauton's media and fluorescence data of 10⁴ cells per assay was acquired using a BD FACS Verse instrument. Experiments were repeated twice in duplicates, statistical significance was analyzed by Student's t-test. Data is represented as an overlay histogram and as a bar graph of MFI values.

For uptake experiments, cells were processed as before, blocking was done with SFM containing 2% BSA for 30 min at 4°C. Subsequently, bacteria were incubated with 10 μg Lf-A647 at 37°C for 2 h. Cells were then washed twice and treated with 0.1% pronase at 4°C for 5 min on ice to remove any residual surface bound proteins. Finally, cells were washed extensively with iron free Sauton's media and fluorescence data of 10⁴ cells per assay was acquired using a BD FACS Verse instrument. Experiments were repeated twice in duplicates, statistical significance was analyzed by Student's t-test. Data is represented as an overlay histogram and as a bar graph of MFI values.

Log phase M.tb H37Ra mCh and M.tb H37Ra GAPDH-mCh cells, (2 × 10⁸ cells / assay) were washed twice with Sauton's media without iron followed by incubation with the same media for 2 h at 37°C. Cells were then washed and incubated for a further 1 h at 37°C with media containing 50 μg of Lf-⁵⁵Fe in 100 μl Sauton's media without iron. Cells were washed thrice with iron-free media, centrifuged, and resuspended in 3.0 ml of scintillation fluid. The radioactivity associated with the cells was measured using a beta counter (Perkin Elmer), counts were normalized to cell number. Statistical significance was estimated by Student's t-test (n = 6) (Boradia et al., 2014).

Log phase M.tb H37Ra mCh and M.tb H37Ra GAPDH-mCh cells, (2 × 10⁸ cells/assay) were washed and blocked with 2% BSA followed by incubation with 20 μg Lf-FITC in 100 μl PBS, for 2 h at 4°C. Samples were fixed in 4% buffered paraformaldehyde for 20 min and FRET analysis was carried out by the acceptor photobleaching method on a Nikon A1R confocal microscope as described previously (Raje et al., 2007). The intensities of a total of 70 regions of interest from multiple, randomly selected fields were measured before and after photobleaching, and FRET efficiency was calculated (Boradia et al., 2014). Statistical analysis was done by Student's t-test.

M.tb H37Rv and M.tb H37Rv ΔmbtB::hyg strains were cultured to log phase, and 1 × 10⁸ cells were used per assay. Cells were washed with phosphate-buffered saline (PBS) and blocked for 1 h at 4°C with 2% BSA in SFM (serum free media). Cells were then incubated with Lf-A647 (10 μg per 100 μl of SFM) alone or in the presence of 200-fold excess unlabeled human Lf at 4°C for 2 h. Finally, cells were washed extensively with SFM and fluorescence data of 10⁴ cells per assay was acquired using a BD FACS Accuri instrument. Experiments were repeated thrice in duplicates. Statistical significance was analyzed by Student's t-test.

Cells (1 × 10⁸ bacilli/assay) were blocked with SFM containing 2% BSA for 30 min at 4°C. Subsequently, bacteria were incubated with 10 μg Lf-A647. After incubation at 37°C for 2 h, cells were treated with 0.1% pronase at 4°C for 5 min to remove any residual surface bound proteins. Cells were washed extensively with SFM and fluorescence data of 10⁴ cells per assay was acquired using a BD FACS Verse instrument, experiments were repeated thrice in triplicates. Statistical significance was analyzed by Student's t-test.

The human macrophage cell line, THP-1, was utilized as an infection model. To study trafficking of Lf to bacteria resident within macrophages, THP-1 cells were plated into Matek glass bottom petri dishes (3 × 10⁵ cells per dish). Cells were activated with 25 ng/ml PMA (Sigma) for 24 h and rested for an additional 24 h. Cells were then shifted to antibiotic-free RPMI media containing 10% fetal calf serum and infected with bacilli at a ratio of 1:20 (THP-1: bacteria) using log phase cultures of M.tb H37Rv-GFP, M.tb H37Ra mCherry, or M.tb H37Ra GAPDH-mCherry. After 6 h, cells were washed with serum-free media (SFM) to remove non-phagocytosed bacteria. A second wash with SFM was carried out after 24 h of infection and cells were shifted to SFM for 30 min at 37°C. Cells were then washed and incubated with 10 μg of Lf-A647 in 100 μl SFM media at 4°C for 30 min. Subsequently, for internalization of bound Lf cells were shifted to 37°C, 5% CO₂ for 1 h. After extensive rinsing with SFM, cells were fixed with 2% paraformaldehyde for 15 min and imaged on a Nikon A1R confocal microscope using a 60X oil immersion objective and an aperture of 1 airy unit.

THP-1 cells were plated and processed for infection with log phase cultures of M.tb H37Ra-GFP and incubation with Lf-A647 as described above. Subsequently cells were imaged live on a Nikon A1R confocal microscope equipped with an Okolab bold line water-jacket cryo topstage CO₂ incubator system (Okolab Italy).

Cells (2 × 10⁹ bacilli per sample) were blocked with SFM containing 2% BSA for 30 min at 4°C. Subsequently, bacteria were incubated with 1 mg of Lf-conjugated with 20 nm gold particles for 2 h at 37°C. Subsequently cells were treated with 0.1% pronase at 4°C for 10 min to remove any residual surface bound proteins. Cells were then fixed, embedded in epoxy resin and ultrathin sections cut for visualization in TEM essentially as described previously (Boradia et al., 2014).

Both mutant forms of the protein were expressed and purified as described previously for wt rGAPDH (Boradia et al., 2016). Purification was confirmed by western blotting, detection was done after incubation with Mouse anti-His (Sigma) (1:3,000) for 1 h followed by incubation with anti-Mouse IgG HRP (Sigma) (1:8,000) for 1 h. The enzyme activity of wt rGAPDH, rGAPDH(N142S), and rGAPDH(P295L) purified from cytosol fraction was studied by measuring the increase in the absorbance at 340 nm due to oxidative reduction of NAD⁺ to NADH. The reaction mixture containing 200 μl of enzyme assay buffer (50 mM HEPES, 10 mM sodium arsenate, and 5 mM EDTA, pH 8.5), 1 mM NAD⁺ and 2 mM glyceraldehyde-3-phosphate (G-3-P) was added to wells containing 100 ng of each purified enzyme at 25°C. Enzyme activity was measured at 340 nm for 5 min on a Tecan Infinity M200 multimode microplate reader. Negative controls were set up without the specific substrate G-3-P and their values were subtracted from the final absorbance. The experiment was repeated thrice in triplicates. The enzymatic activity of wt rGAPDH was taken as 100% and data was plotted as % residual activity ± SD. Statistical analysis was done using Student's t-test.

Lf (3 μg), BSA (5 μg), wt rGAPDH (1 μg), rGAPDH (N142S) (1 μg), or rGAPDH (P295L) (1 μg) were resolved on 10% SDS-PAGE and then transferred to nitrocellulose membrane. The membrane was then blocked with 5% BSA and probed with 10 μg/ml of either wt rGAPDH, rGAPDH(N142S) or rGAPDH(P295L) for 2 h, followed by incubation with mouse anti-His (Sigma) (1:3,000) for 1 h and anti-mouse IgG HRP (Sigma) (1:8,000) for 1 h. Finally, blots were washed and developed with TMB/H₂O₂; the experiment was repeated three times.

Recombinant M.tb H37Rv GAPDH, wild type (wt rGAPDH) was prepared as described previously (Boradia et al., 2016). Lf, BSA (3 μg each) and wt rGAPDH (400 ng) were run on a 10% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was probed with 10 μg/ml of wt rGAPDH for 2 h, followed by incubation with rabbit anti-GAPDH (1:1,000) for 1 h and anti-rabbit IgG HRP (1:16,000) for 1 h; blots were developed with TMB/H₂O₂, experiments were repeated thrice.

The affinity of the GAPDH and Lf interaction was estimated using a plate based solid phase binding assay. Polystyrene wells were coated overnight at 4°C using 2 μg/well of either wt rGAPDH, rGAPDH(N142S) or rGAPDH(P295L) mutant proteins in PBS (pH 7.4) and blocked using 5% cold fish skin gelatin prepared in PBS for 4 h at room temperature. GAPDH coated wells were then incubated at 25°C for 2 h with increasing concentrations (7.5−1,000 nM) of biotinylated Lf in PBS containing 1% fish skin gelatin. Control wells were incubated with buffer alone. Extensive washing with PBST was done out to remove any unbound Lf. Bound Lf was detected using primary antibody Mouse anti-Biotin (1:5,000), secondary antibody anti-Mouse IgG HRP (1:20,000) followed by the addition of TMB-H₂O₂ for development of color. Data was plotted as absorbance at 650 nm vs. concentration of biotinylated Lf after subtracting OD values of control wells. Experiments were performed thrice (with triplicates) and data was plotted for non-linear fit, one-site specific binding using Graph Pad Prism®.

Initial evolutionary conservation sequence analysis to identify functionally and structurally important residues in M.tb GAPDH were performed by the ConSurf Program (Ashkenazy et al., 2010, 2016). The primary sequence of wild type, N142S and P295L mutants as well as M.tb GAPDH modeled structure were considered. For query sequences and structure the CSI-BLAST (Context-Specific Iterated-Basic Local Alignment Search Tool) homolog search algorithm were used with 0.00001 as E-value cutoff, set at a maximal 95% identity and minimal identity of 35% between sequences for homology searches. The protein database UNIREF-90 (http://www.uniprot.org/help/uniref) was utilized; all other parameters were kept at default values for calculation of conservation scores. The primary goal was to reveal the highly conserved regions and functionally and structurally important residues in M.tb GAPDH.

GAPDH is a homo-tetramer composed of four identical 36 kDa subunits. The structure of GAPDH alongwith its associated cofactor NAD⁺ has been reported for other homologous organisms. The sequence of M.tb H37Rv GAPDH was retrieved from NCBI (GenBank: CAB09248.1), the protein sequence is composed of 339 amino acids. The sequence was analyzed by NCBI protein BLAST search against Protein Data Bank (PDB) in order to get a template with a suitable identity. Tetrameric GAPDH from Bacillus stearothermophilus (PDB ID: 1GD1) was identified as the best template and was utilized for homology modeling of GAPDH using Modeller9v program (Webb and Sali, 2014).

GAPDH homo-tetramer assembled from four identical 36 kDa subunits was done using superimposition tool of Chimera program (Pettersen et al., 2004; Meng et al., 2006). The template (PDB ID: 1GD1) was assembled into its homo-tetrameric structure, each unit of modeled M.tb GAPDH was then superimposed upon this template. The assembled model was energy minimized to correct the unfavorable bond lengths, bond angles, torsion angles, and contacts. Subsequently, the final model was verified and validated using RAMPAGE Ramachandran plot analysis server (http://mordred.bioc.cam.ac.uk/~rapper/rampage.php) and native protein folding energy assessment by PRoSA program (https://prosa.services.came.sbg.ac.at/prosa.php). The position of the two mutations was analyzed in context of the GAPDH tetramer and its proximity to the NAD⁺ binding site.

Two selected mutations (N142S and P295L) were experimentally tested for the two functions attributed to GAPDH i.e., (i) enzyme activity and (ii) ability to bind Lf. Of these Asparagine 142 is a conserved residue and mutation to serine resulted in a loss of enzyme activity. However, both mutants retained their ability to bind Lf in far-western blots. Both mutants were explored to predict the effect of these alterations on protein stability and proximity to substrate binding site. The effect of mutations on protein stability were analyzed using DUET (Pires et al., 2014), mCSM and Site Directed Mutator (SDM) (Worth et al., 2011).

Flow cytometry clearly demonstrated the binding of Lf to M.tb H37Ra cells which was significantly inhibited (p < 0.0001) in the presence of 100-fold excess of unlabeled Lf, indicative of a specific interaction (Figures 1A,B). The binding of Lf-gold particles conjugated particles to M.tb H37Ra was confirmed by TEM analysis (Figures 1C–E).

M.tb H37Ra strains overexpressing GAPDH-mCherry (GAPDH-mCh) or mCherry (mCh) alone were utilized to confirm whether Lf iron is GAPDH dependent. The overexpression of GAPDH in M.tb H37Ra resulted in enhanced binding (Figures 2A,B) and uptake of Lf when compared to control cells that over express mCherry alone (p < 0.0005) (Figures 2C,D). A correlated increase in iron uptake was also evident in the M.tb GAPDH-mCh strain as compared to the mCh strain (Figure 2E).

To confirm that GAPDH and Lf interact on the bacterial cell surface we carried out acceptor photobleaching Förster resonance energy transfer (FRET) analysis using M.tb H37Ra cells expressing GAPDH-mCh or mCh alone. This assay is based on the principle of two interacting partners which are fluorescently tagged. The fluorophores are selected such that the emission spectra of the donor overlaps with the absorption spectra of the acceptor. In the current study if Lf-FITC and GAPDH-mCherry interact, when excitation is done with the wavelength specific for Lf-FITC a strong fluorescence of mCherry is evident. When mCherry is destroyed (by photobleaching) an increase in donor signal is visible because the acceptor is no longer available to parasitize the light emitted from Lf-FITC (Kenworthy, 2001). In the present study a significant increase in donor (Lf-FITC) fluorescence intensity was observed upon photobleaching of acceptor (mCh) in the M.tb H37Ra GAPDH-mCh strain (Figures 2F,H) as compared to the strain expressing mCh alone (Figures 2G,H). The occurrence of FRET between the GAPDH and Lf indicates that they are at proximity of 1–10 nm (Kenworthy, 2001).

Both strains demonstrated cell surface binding of human Lf at 4°C that was competitively inhibited by the presence of 200X unlabeled Lf (Figures 3A,B). Lf binding was comparable in wild type and siderophore knockout strains (Figure 3C). Internalization of labeled Lf was also evident in both strains after incubation at 37°C (Figure 3D) indicating that GAPDH mediated Lf uptake is siderophore independent.

Bacilli were analyzed by TEM to assess the internalization of Lf-gold conjugated particles, which were observed within the cell cytosol (Figures 3E–G).

THP-1 cells were infected with either (i) M.tb H37Ra mCh (ii) M.tb H37Ra GAPDH-mCh or (iii) M.tb H37Rv-GFP strains (Figures 4A–C respectively). The trafficking of Lf-A647 to the intraphagosomal bacilli was assessed by confocal microscopy. In all cases the co-localization of Lf-A647 and bacilli was observed, live cell imaging of infected cells confirmed the endocytosis and subsequent co-localization of Lf with intraphagosomal M.tb H37Ra-GFP (Movie 1).

Both proteins were expressed using M.tb H37Ra as a host strain. The proteins were purified using Ni-NTA affinity chromatography and purity confirmed by western blotting with mouse anti-His antibody (Figures 5A,B).

The N142S mutant showed a significant loss of activity (p < 0.0001), only 45% enzyme activity was retained as compared to the wild type enzyme. In contrast the P295L mutant protein demonstrated enzyme activity comparable to wt rGAPDH (Figure 5C). The GAPDH-Lf interaction was confirmed in vitro by far western blotting using recombinant M.tb rGAPDH (rGAPDH) (Figure 5D). Resolved Lf was able to capture rGAPDH as evident by far western blotting, rGAPDH, and BSA were utilized as positive and negative controls respectively. The N142S, P295L mutant proteins also retained their ability to bind Lf in far western blots (Figures 5E,F). ELISA was carried out using 125 nM Lf to obtain quantitative data, no significant difference was observed between the three proteins (Figure 5G).

Lf binding to rGAPDH and mutants was observed to steadily increase with increasing concentration of Lf with saturation at 500 nM (Figure 5H). The K_D value of GAPDH-Lf interaction was estimated to be 31.7 ± 1.68 nM, a 5-fold higher affinity as compared to M.tb GAPDH and Tf (160 ± 24 nM). No significant difference in the affinity values were evident between wt and mutant proteins values. The N142S and P295L mutants were 38.22 ± 3.08 and 37.74 ± 2.63 nM respectively (Figure 5H).

The M.tb GAPDH sequence was analyzed for its evolutionary conservation, it was observed that key active site residues as well as other surface exposed residues are highly conserved. Since it is known that GAPDH from multiple species binds to Tf (Modun and Williams, 1999), there is a high probability that the conserved regions (including active sites, co-factor binding site, and surface regions) may contribute to the interaction between Lf and Tf. Consurf server based evolutionary conservation analysis was carried out for the wt GAPDH sequence and the two point mutations N142S and P295L (http://consurf.tau.ac.il/2016/). Analysis of Asparagine 142 (N142) revealed that it is a highly conserved and exposed functional residue while Proline at 295 position is an exposed but variable residue (Figure 6A).

The comparative modeling of M.tb GAPDH was performed, using highly identical template of GAPDH from B. stearothermophilus (PDB ID: 1GD1). The two sequences demonstrate a sequence identity of 60%. The modeled 3D structure of GAPDH was validated by the RAMPAGE Ramachandran plot analysis server (http://mordred.bioc.cam.ac.uk/rapper/rampage.php). The quality of protein folds of GAPDH homology model was evaluated using ProSA protein structure analysis web server (https://prosa.services.came.sbg.ac.at/prosa.php). Modeled 3D structure aligned with the template (PDB ID: 1GD1) and assembled structural model was developed using UCSF Chimera software (https://www.cgl.ucsf.edu/chimera/), the RMSD score was 0.238 (Figures 6B,C).

Although, in silico analysis of the mutant N142S suggests that this mutation may result in a non-significant structural change, since Serine at position 142 also shows a similar characteristic to the wild type residues N142, experimentally the mutation resulted in a loss of enzyme activity. Several computational methods have been developed for predicting the stability of mutant proteins based on sequence, structure, and Gibbs free energy estimation, this combined feature based approach provides better prediction of changes due to mutations (Kulshreshtha et al., 2016). In the current study, two different methods were used to predict the stability change of mutated proteins, predictions also assessed the sequence conservation. A combined sequence conservation and protein modeling study indicates Asparagine (N142) is a highly conserved and exposed residue that is located in a flexible loop region of GAPDH. Predicted stability change energies in the mutant (ΔΔG) indicate destabilization (Table 1). Structural analysis indicates that in M.tb GAPDH, the Glycine-Valine-Asparagine tripeptide sequence (at positions 140, 141, and 142 respectively) is also highly conserved in several GAPDH homologs. Additionally, it was observed that Asparagine 142 also has hydrogen bonding interactions with another highly conserved tripeptide region comprised of residues 136 to 138 (Figures 6, 7C). This tripeptide is composed of the sequence Threonine136-X-Valine138 where X indicates a variable residue. In M.tb GAPDH the intervening residue is Isoleucine at position 137 (Figure 6). Replacement of Asparagine with serine results in a loss of hydrogen bonding with Threonine 136 (Figure 7D). Computational modeling of the N142S mutant revealed conformational changes in this region during loop refinement, suggesting that this mutation may alter the stability of the molecule or its interaction with a helix that lies in close proximity (Figures 7A,B). While biochemical assays confirm a loss of enzyme activity, detailed analysis by co-crystallization, molecular modeling, extensive dynamic simulation, and mutagenesis would be essential to ascertain the relevance of these residues (if any) in the GAPDH-Lf/Tf interaction. In contrast the Proline 295 residue occurs in a variable loop region adjoining a highly conserved helix, mutation to Leucine does not result in a structurally significant change as confirmed by experimental data (Figures 7E,F).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.