Oadc enrichment

Mycobacterium tuberculosis Erdman (American Type Culture Collection 35801) was the parental strain used for all experiments. The fluorescent reporters smyc′::mCherry, hspx′::GFP/smyc′::mCherry, and hsp60′::GFP were previously described (Abramovitch et al., 2011 (link); Dhandayuthapani et al., 1995 (link); Sukumar et al., 2014 (link); Tan et al., 2013 (link)). Bacteria were grown at 37°C to mid-log phase in MiddleBrook 7H9 broth supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC Enrichment; Becton, Dickinson and Company), 0.2% glycerol, and 0.05% tyloxapol (Sigma-Aldrich). BCG (Pasteur) was grown as described above. Hygromycin B (50 mg/ml) was used as a selection marker for the fluorescent strains. For mice infection, aliquots were frozen in 10% glycerol, titered, and stored at −80°C until use.

Remnant pellets of processed sputum samples were resuspended in 400 μl of Middlebrook 7H9 broth supplemented with OADC Enrichment (Becton, Dickinson and Company, Sparks, MD, United States) and PANTA (Becton, Dickinson and Company, Sparks, MD, United States) and divided in four 100 μl aliquots. In two aliquots, 4 μg/ml RIF was added and the other two were kept as control. Samples were recovered for 48 or 96 h and infected overnight with 5 μl of a stock of mCherry_bombϕ (10¹⁰–10¹¹ PFU/ml). After that, samples were fixed with 2% paraformaldehyde for 2 h at room temperature, centrifuged and the pellet was resuspended in 10 μl of phosphate buffered saline (PBS) and examined by fluorescence microscopy using 1,000× magnification. The criterion for detection was the presence of at least one fluorescent bacillus per 100 fields in the control samples and for determination of RIF resistance was the same in RIF treated samples. We also compared the results using the criterion of two fluorescent bacilli per 100 field but we did not observed significantly differences between both criteria so we opted for the first one. Detection results using mCherry_bombϕ were compared with growth and colony counting (CFU) in LJ media and determination of RIF susceptibility with the proportion method (Canetti et al., 1963 (link)).

Mtb H37Rv (ATCC) was initially grown in Middlebrook 7H9 medium (7H9) supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD, United States), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v; Sigma) prior to inoculation in a MM consisting of KH₂PO₄ 1 g/l, Na₂HPO₄ 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, CaCl₂ 0.5 mg/l, ZnSO₄ 0.1 mg/l, with or without Tyloxapol 0.05% (v/v), containing 0.1% (v/v) glycerol, pH 7.0. Cultures were grown in 7H9 at 37°C until they reached an OD of 0.6 and then washed in in phosphate-buffered saline (PBS) + 0.05% Tyloxapol and transferred to MM. Low iron minimal medium (LIMM) was used for culture under iron-controlled conditions. To remove metal ions MM was treated with 5% Chelex-100 (Bio-Rad, Hercules, CA, United States) for 24 h, at 4°C with gentle agitation. Chelex-100 was removed by filtration through 0.22 μm filter (Millipore, Burlington, MA, United States) and the medium was supplemented with sterile ZnCl₂ 0.5 mg/l, of MnSO₄ 0.1 mg/l, and of MgSO₄ 400 mg/l. The amount of residual Fe in this medium, determined by atomic absorption spectroscopy is ∼1 μM. High iron MM (MM) was prepared by supplementing LIMM with 50 μM FeCl₃.

M. bovis BCG Pasteur and Mtb H37Rv were grown in minimal medium (MM) [KH₂PO₄ 1 g/l, Na₂HPO₄ 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, MgSO₄×7 H₂O 0.5 g/l, CaCl₂ 0.5 mg/l, ZnSO₄ 0.1 mg/l, 0.1% (v/v) glycerol, and with or without Tyloxapol 0.05% (v/v; Sigma), pH 7.0] or in Middlebrook 7H9 supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v) for 14 days in a 5% CO₂ incubator at 37°C. Mtb lineages were a gift from Sebastien Gagneux. Mtb lineages were systematically grown in MM supplemented with 30 mM pyruvate.
Recombinant Ag85b was obtained from AERAS Tb vaccine Foundation (Rockville, MD). Recombinant PA (Protective Antigen from Bacillus anthracis) was obtained from David Axelrod Institute, Albany, NY). The 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) and the other reagents used during the conjugation reaction were purchased from Sigma. The CS-35 monoclonal antibody recognizing LAM and AM, was obtained from BEI resources (Manassas, VA). The monoclonal antibody 9d8 specifically recognizes mycobacterial capsular AM [10 (link), 54 (link)]. The monoclonal antibody 24c5 specifically recognizes mycobacterial capsular α-glucan [55 (link)]. Alhydrogel was purchased from InvivoGen (San Diego, US).

All bacterial strains were derived from Mtb strain Erdman and cultured at 37°C. Liquid medium: Middlebrook 7H9 (Difco) supplemented with 0.5% albumin, 0.2% glucose, 0.085% NaCl, 0.5% glycerol, and 0.02% Tyloxapol. Solid medium: Middlebrook 7H11 (Difco) supplemented with 10% OADC enrichment (Becton Dickinson) and 0.5% glycerol. Aliquots were stored in 15% glycerol at −80°C and used once. All strains were transformed with a plasmid integrated at the chromosomal attB site to allow constitutive expression of the fluorescent protein tdTomato under the control of the hsp60 promoter. Wild-type (WT) refers to the Erdman strain constitutively expressing tdTomato. The 5’Tn::pe35 (ESX-1 deficient) strain was generated using transposon mutagenesis (Chen et al., 2013 (link)).