Animal experimentations were performed inside the biosafety facilities of the National Institute of Agricultural Technology (INTA), Argentina, in compliance with the regulations of Institutional Animal Care and Use Committee (CICUAE) of INTA (file number: 31-20-12). CICUAE’s members: Florestán Maliandi (President), Alejandra Romera (Secretary), Marisa Farber, Analía Berinstein, Pablo Chacana, Gabriel Pinto, Bibiana Brihuega, Gisella Marcoppido, Verónica Maldonado May, Lucas Vagnoni, Osvaldo Zabal and Luis Samartino (Vocals).
Bacterial strains and culture media
All cloning steps were performed in Escherichia coli HB101. E. coli were grown either in Luria-Bertani (LB) broth or on LB agar. M. tuberculosis strains were grown in Middlebrook 7H9 medium supplemented with albumin 0.5%, dextrose 0.4%, and glycerol 0.5% (M7H9-AD-G) and either Tween 80 0.05% or Middlebrook 7H11, supplemented with albumin, dextrose and glycerol. When necessary, either 50 μg/ml hygromycin or 20 μg/ml kanamycin was added to the media.
Construction of M. tuberculosis Δmce2R mutant and complemented strains
A mutant strain of M. tuberculosis carrying a chromosomal deletion encompassing the bases 137–617 of the mce2R (Rv0586) gene was obtained by using the gene knockout system described by Bardarov . Briefly, two DNA fragments of approximately 1 kb flanking the 5′ and 3′ regions of mce2R were obtained by PCR using M. tuberculosis H37Rv genomic DNA as template and the following sets of primers: Regionup-up (tctagaccgtacaactcgatcaat)/Regionup-low (tctagaactccgagcaactcagcc) and Regionlow-up (actagtatctgctcaggtgatccc)/Regionlow-low (actagtacgccgatcgtggtcaac). Flanking arms were directionally cloned into XbaI and SpeI sites of cosmid pYUB854 . The recombinant cosmid was digested by PacI and ligated to PacI-digested concatemerized DNA of phage phAE87. To generate each specialized transducing phage, the PacI-digested recombinant cosmid was used to replace cosmid pYUB328 in phAE87 an in vitro λ-packaging reaction (GIGAPackII, Stratagene). After transducing E. coli HB101 and plating the transductants on selective media containing hygromycin. Phasmid DNA was prepared from the pooled antibiotic-resistant transductants and electroporated into M. smegmatis mc2155. Transductants were grown at the permissive temperature of 31°C to allow phage replication, and then transducing phages were prepared from isolated plaques as previously described . Transducing phages, carrying the mutated allele of mce2R were used to infect M. tuberculosis H37Rv as previously described . Infected mycobacteria were plated onto media containing hygromycin at the restrictive temperature of 37°C. Colonies that appeared after 25 days of culturing were analysed by PCR for the presence of the deletion in the mce2R gene. Only one clone showed a 480-bp deletion from mce2R and was referred to as MtΔmce2R. Deletion of mce2R in MtΔmce2R strain was confirmed by PCR analysis using two sets of primers: one set that hybridises inside mce2R (WT-forward: gatctgttgccccgattgt/WT-reverse: tctacgatcgaaccggcgct), and the other that hybridises at approximately 1000 bp from the 5′ ends of both mce2R and inside the hygromycin resistance gene (KO-forward [Low new2R] acgtcagcttcagccagagt, KO-reverse [5′hygro-reverse]: tcagcaacaccttcttcacg).
In order to complemente the mutant phenotype, a fragment containing mce2R gene was amplified by PCR using the primers up mce2r pw16 (catatgatctgttgccccgattgttgt) and low mce2r pw16 (catatgcattgccgactcgcct), and cloned into pW16 to produce plasmid pW16mce2R. This plasmid was used to transform the M. tuberculosis MtΔmce2R strain by electroporation to produce the complemented strain MtΔmce2RComp.
The experimental BALB/c model of progressive pulmonary tuberculosis has been previously described in detail . Briefly, bacillary suspensions were adjusted to 1.25 × 105 viable cells in 100 μl phosphate buffer saline (PBS). Each animal was anesthetized and intratracheally inoculated with M. tuberculosis strains. Infected mice were kept in cages fitted with microisolators connected to negative pressure. Groups of 15 mice were each infected with the different M. tuberculosis strains. The inoculum doses were determined by enumerating the CFUs recovered from the lungs of five mice per infection strain 24 h post-infection. Five mice per group were killed at 1, 26 and 35 days after infection and lungs removed and homogenized. Four dilutions of each homogenate were spread onto duplicate plates. This experiment was repeated twice with similar results. Animal experimentations were performed inside the biosafety facilities of the National Institute of Agricultural Technology (INTA), Argentina, in compliance with the regulations of Institutional Animal Care and Use Committee (CICUAE) of INTA. Student’s t test was used to determine significant differences between groups.
M. tuberculosis strains were cultivated until exponential growth phase, pelleted, washed twice in PBS and re-suspended in RPMI medium to a multiplicity of infection (m.o.i.) of 5. Clumps were removed by ultrasonic treatment in a water bath followed by a low speed centrifugation for 2 min. Macrophages were seeded into 24 well tissue culture plates at 80% confluence and infected for 1 hour (uptake). Afterwards, cells were washed and incubated in full medium for another 2 hours (chase).
Inmunofluorescense and confocal microscopy
For indirect immunofluorescence, M. tuberculosis strains treated as described above, were covalently stained with FITC (isomer I; Sigma. FITC solution was prepared 20 mg/ml in DMSO). Briefly, 1 × 109 bacteria were washed twice with 0.1 M buffer Na2CO3/NaHCO3 (pH 9) and suspended in 1 ml of the same solution. FITC was added to a final concentration of 1 mg/ml and incubated in the dark for 2 h at 37°C. Bacteria were washed gently with PBS until unbound colorant was eliminated, and used to infect J774 macrophages as was described above. Infected cells were fixed with 3% paraformaldehyde solution in PBS for 20 min and quenched by incubating with 50 mM glycine solution for 10 min. Then, cells were permeabilized with 0.05% saponin in PBS containing 0.2% BSA for 15 min, and incubated with the primary anti-LAMP-2 (ABL-93, DSHB) antibodies diluted 1:50 in PBS. anti-LAMP-2 antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242. Secondary antibodies anti-Rat Cy5-conjugated (Jackson Immuno Research Labs Inc.) was used diluted 1:600 in PBS. Each step with antibodies was incubated for 1 hour. Cells were mounted with Dako mounting media (Dako, Denmark) and analysed by confocal microscopy using a Leica SP5 AOBS confocal microscope (Leica Microsystems, Germany). Internalization of the mycobacteria was followed through the fluorescence of green FITC and the LAMP-2 association to mycobacterial phagosomes was counted in at least 50 cells using Fiji/ImageJ program (U.S. National Institute of Health, Bethesda, Maryland, USA). The analysis was performed for duplicates in three-four independent experiments. Statistical determinations were made using t test.
DNA-free RNA was extracted from 50 ml mid-exponential-phase cultures of M. tuberculosis as described by Santangelo et al. (2002) .
Prehybridisation, hybridisation, and washing steps were performed as described previously [13, 19]. Microarrays were hybridised with a combination of Cy3-cDNA generated from genomic DNA of M. tuberculosis H37Rv and Cy5-cDNA obtained from total RNA of either M. tuberculosis H37Rv or MtΔmce2R.
Eight sets of microarray data, consisting of eight biological replicates (cells from independent cultures), were produced for each M. tuberculosis strain.
The microarrays were scanned using an Affymetrix 428 scanner and fluorescent spot intensities were quantified using BlueFuse for Microarrays v3.2 (BlueGnome Limited, http://www.cambridgebluegnome.com). For each spot, background fluorescence was subtracted from the average spot fluorescence to produce a channel specific ratio.
Data processing and statistical analysis
Log2 Cy5:Cy3 (test:control) ratios were used for subsequent calculations. Within each microarray, block median normalisation, excluding control and empty spots, was carried out using the BlueFuse software. Median absolute deviation using Mathematica 5.2 (Wolfram Research Inc.) was applied to bring the histograms of all microarrays into the same scale. Technical replicates were averaged. Differentially expressed genes between the strains were detected by applying t-tests with a Benjamini and Hochberg adjusted p-value correction.
RT-qPCR reactions were performed as described by Santangelo et al. [13, 20] using DNA-free RNA (1 μg) extracted from mid-exponential growth-phase cultures and specific primers. Relative quantification was performed by using sigA as a reference gene and a subsequent analysis for statistical significance of the derived results was performed by using the Pair Wise Fixed Reallocation Randomization test . The mean value of PCR efficiency for the primers (Additional file 2: Table S2) was 92% to 100%. These values were calculated using both the classical dilution curve and slope calculation (E = 10 [−1/slope] − 1)  and an estimation by absolute fluorescence increase .