Antibodies and reagents
We used rabbit anti-ESAT-6 antibody (Abcam, Cambridge, MA), mouse anti-His6X antibody (Sigma-Aldrich, St. Louis MA), anti-FLAG antibody (Sigma-Aldrich, St. Louis MA), HRP conjugated anti-mouse IgG (Bio-rad, Hercules, CA), anti-rabbit FITC conjugated secondary antibody (Jackson-ImmunoResearch, West Grove, PA) and anti-mouse Texas Red secondary antibody (Jackson-ImmunoResearch, West Grove, PA) for in vitro studies. For in vivo studies, anti-CD4 (clone: GK1.5)-FITC, -PerCP-Cy5 or -APC, anti-CD8 (clone: 53–6.7)-FITC, -PerCP-Cy5 or -APC, anti-CD44 (clone: IM7)-APC, anti-Brdu (clone: Bu20a)-PE, anti-CD11b (clone: M1/70)-APC, anti-CD11c (clone: N418)-APC, 7AAD, anti-IFN-γ (clone: XMG1.2)-APC, anti-IL-17 (clone: TC11-18H10.1)-PE, anti-IL-4 (clone: 11B11)-PE, anti-IL-6 (clone: MPS-20 F3)-PE, anti-IL-12 (clone: C15.6)-PE, anti-IL-22 (clone: Poly5164)-PE, anti-IL-10 (clone: JES5-16E3)-PE, anti-IL-9 (clone: MH9A4)-PE, anti-TNF-α (clone: MP6-XT22)-PE, (all from Biolegend, USA) and anti-CD69 (clone: H1.2 F3)-PE (from eBiosciences, USA) were used.
BALB/c female mice at 6–8 weeks of age were used throughout this study, following institutional ethical committee guidelines. All animal experiments were conducted in accordance with guidelines approved by the Institutional Animals Ethics Committee of ICGEB, New Delhi, India and Department of Biotechnology (DBT), Government of India. Mice were housed under barrier conditions in a Biosafety Level III laboratory.
Bacterial two-hybrid studies
Bacteriomatch™ system was used as described earlier [16, 17]. Two colonies each from HCL2pTRGnn + ESAT-6pBTnn, positive and negative, were grown in cultures and patched on X-gal indicator plate. Interactions were validated by liquid β-Galactosidase assay and compared with well-established mycobacterial interaction between ESAT-6 and CFP10 (ESAT-6pBTnn + CFP10pTRGnn).
In vitroFar-Western Dot Blot analysis
For Far-Western Dot Blot assay, ESAT-6-FLAG was purified by ion-exchange chromatography. Briefly, E. coli BL21 (DE3) cells harboring flagesat6pET28a were subjected to IPTG induction and cells were harvested, lysed in denaturing condition. Clear supernatant was allowed to bind on DEAE-sepharose (Amersham) followed by washing with lysis buffer and the resin bound proteins were eluted using a gradient of NaCl from 0 M to 1 M, prepared in lysis buffer. Purified protein was refolded by sequential dialysis against PBS with decreasing concentration of urea. The interaction between ESAT-6-FLAG and HCL2-His6X (commercially synthesized from GenScript, Hong Kong) was determined using a protocol reported previously . An unrelated protein ED3-His6X (sequence given in Additional file 1: Figure S1) was used as a negative control. CFP10-His6X and ESAT-6-FLAG were used as positive controls.
Cloning of HCL2 in pMTSA vector
The ORF encoding HCL2 peptide was amplified from original lung cDNA library clone using 5’-AAGGATCCTACGTAAGAATTCGGCACGAG-3’ and 5’-AAGGATCCTACGTAGAAAA ATCCTGCGAAGAAAA-3’ as forward and reverse primers respectively. The gene was cloned in a SnaBI site in an arabinose-inducible three-hybrid vector as previously described . Expression of HCL2-His6X was analyzed on Tricine-SDS PAGE and Western Blotting was done using Anti-His6X antibodies.
Bacterial Three-Hybrid and arabinose gradient liquid β-Galactosidase assay
Bacterial Three-Hybrid studies were carried out as described earlier . To study the correlation between disruption of the ESAT-6:CFP10 interaction and in vivo expression levels of HCL2, an Arabinose gradient liquid β-Galactosidase assay was performed. The experiment was carried out in triplicates by varying L-arabinose concentration ranging from 0 to 1%. Western Blotting was done with increasing concentration of L-arabinose (0-1%), to show the gradual increase in HCL2-His6X induction corresponding to decrease in β-Galactosidase activity.
HCL2 cloning in a mycobacterial shuttle vector
The ORF expressing HCL2 peptide was cloned in mycobacterial constitutive expression vector pVV16 and pVVGFPHis6X and used to electroporate H37Rv electro-competent cells, as described earlier .
Effect of HCL2 on M. tuberculosisgrowth
We examined the effects of HCL2 peptide on M.tb growth by using two methods. First, by electroporating HCL2pVV16 (H37Rv/HCL2; strain expressing HCL2-His6X) and secondly by adding HCL2-His6X peptide to the culture exogenously at a final concentration of 15 μg/ml (H37Rv + HCL2). HCL2 was cloned in mycobacterial constitutive expression vector pVV16 as described earlier . Cultures were inoculated in triplicates each for H37Rv/HCL2, H37Rv + HCL2, H37Rv/pVV16 (strain having only plasmid control), ΔRD1, H37Rv/GFP, H37Rv + DL1 and H37Rv and assessed spectrophotometrically for 18 days at 630 nm. H37Rv was added with equivalent amount of Milli-Q water used to dissolve the peptide HCL2-His6X. A higher concentration of HCL2-His6X was not used due to precipitation of the peptide at concentrations > 15 μg/ml. Strain lacking RD1 region (ΔRD1; kind gift of Prof. David R. Sherman), H37Rv expressing GFP (H37Rv/GFP), and addition of an unrelated peptide (sequence given in Additional file 1: Figure S1) DL1 (H37Rv + DL1) were used as controls. Another ESAT6 binding peptide, SL3, from our previous studies  was also analyzed for its effects on mycobacterial growth during this study (unpublished results).
Effect of HCL2 on cellular and colony morphology
Electron microscopy was carried out to examine the effect of endogenous or exogenous HCL2 on cellular morphology of M.tb. H37Rv/HCL2, H37Rv + HCL2, H37Rv, ΔRD1 and H37Rv/pVV16 cells were fixed by treating with 2% paraformaldehyde solution and adsorbed on a 300 mesh copper grid and air dried. Samples were stained with 1% uranyl acetate followed by photography using a FEI Tecnai 12 Electron Microscope. H37Rv, H37Rv/pVV16 and ΔRD1 strains were used as controls. In addition, colony morphology of H37Rv/HCL2 strain was observed on 7H11 plates and colony texture was compared with H37Rv.
Infection of THP-1 cells by M. tuberculosisin presence of HCL2 peptide
Approximately 12,000/well phorbol-12-myristate-13-acetate (Sigma, USA) activated THP-1 cells in a 96 well flat bottom tissue culture plate were infected with M.tb H37Rv/HCL2 at an MOI of 1:10 using a protocol described previously . Cells were harvested, lysed and plated on 7H11 agar at 0, 24, 48 and 72 hours and CFU counts determined. For effects of exogenous HCL2-His6X (H37Rv + HCL2), peptide was added to RPMI media at a final concentration of 15 μg/ml and this media was added to H37Rv infected tissue-culture plates at 0 hours and changed after every 24 hours till 72 hours. H37Rv, H37Rv/GFP, H37Rv + DL1 and ΔRD1 were used as control strains to infect THP-1 cells. In this experiment, SL3 peptide was also analyzed for its effects on intracellular survival (unpublished results).
Mice infection with H37Rv/HCL2, CFU counts and immunology
BALB/c mice were infected with ~150 CFU of H37Rv and H37Rv/HCL2 using an aerosol chamber. Mice were sacrificed at different time points and cytokine profile and T lymphocytes proliferation were assessed as described earlier . For CFU counts lung and spleen were harvested at different time points and processed as described previously .
All experiments were repeated thrice and in triplicates. Mean values were calculated with standard deviation (STDEV) unless stated otherwise. For all statistical analyses Student's T-test was performed to compare two groups; p < 0.05 was considered significant.