H37ra

Preparation of M.tb (H37Ra, ATCC 25177, Manassas, VA, USA) for PBMC infection was done as described previously [22 (link)]. M.tb suspensions were prepared in Middlebrook 7H9 broth medium supplemented with 10% albumin dextrose catalase (BD Bioscience) and 0.2% glycerol. After a 21-day incubation period at 37 °C on an orbital shaker, M.tb stock suspensions were harvested and concentrations assessed by colony-forming unit (cfu) counts on 7H10 solid agar plates after 21-day incubations. Aliquots were then made and stored at −86 °C until use in in vitro infection experiments.
For PBMC infection experiments, single cell M.tb suspensions were prepared as follows: frozen M.tb stock was thawed, centrifuged for 5 min at 8000× g and re-suspended in complete culture medium. Single bacterial cell suspensions were generated by declumping (5 min. vortexing with 5 sterile 3-mm glass beads) from M.tb stock suspensions. An additional centrifugation step (350× g for 5 min.) was added to remove any remaining M.tb clumps. To generate desired multiplicities of infection (MOI, i.e. the ratios of M.tb to monocytes for in vitro infections) of 1 (MOI1) and 5 (MOI5), percentages of monocytes in PBMC from each study participant were assessed by flow cytometry. Concentrations of M.tb in thawed M.tb stock suspensions were confirmed in each infection experiment by cfu assays.

The following laboratory M. tuberculosis strains were used: H37Ra (ATCC 25177), H37Rv (ATCC 27294), and SS18b (donated by Stewart Cole), and 25 clinical strains from the South African Medical Research Council. Storage and culture conditions for log-phase bacteria, SDB, and intracellular M. tuberculosis were as described in our prior studies (23 (link), 68 (link), 69 (link)). M. tuberculosis SS18b was first cultured in Middlebrook 7H9 broth plus 10% OADC in the presence of 50 mg/liter of streptomycin to achieve log-phase growth, followed by subculture in the same medium but without streptomycin for 14 days. Human-derived THP-1 cells (ATCC TIB-202) grown in RPMI 1640 plus 10% fetal bovine serum FBS (RPMI-FBS) were infected with H37Ra for intracellular experiments in wells and HFS-TB using methods as described previously but with a coincubation period of 3 h.

Mtb strains H37Rv and H37Ra were obtained from American Type Culture Collection (Manassas, VA). LprG null strains were generated from Mtb H37Rv (H37Rv ΔlprG; N. Banaei) and H37Ra (H37Ra ΔlprG; E.T. Richardson) using a specialized transducing phage targeting 571 bp within the lprG gene locus for homologous recombination with a hygromycin resistance cassette. Mtb H37Rv ΔlprG::lprG-Rv1410c was then generated by complementing Mtb H37Rv ΔlprG with the native Rv1411c/1410c operon expressed off the kanamycin-selective integrating plasmid pMV306. Bacteria were cultured in Middlebrook 7H9 broth (Difco, Lawrence, KS) supplemented with 10% albumin/dextrose/catalase (BD, Franklin Lakes, NJ), 0.05% Tween 80 and 0.2% glycerol plus 100 µg/ml hygromycin B (Invivogen, San Diego, CA) for H37Ra ΔlprG and H37Rv ΔlprG or 50 µg/ml kanamycin (Sigma, St. Louis, MO) for H37Rv ΔlprG::lprG-Rv1410c.
To assess bacterial expression of lipoglycans, lysates were prepared from Mtb H37Ra and H37Ra ΔlprG. Bacteria were grown to mid log phase in 50 ml of Sauton's medium, pelleted, suspended in PBS, and sonicated in an ice bath for 40 min in 10-second cycles at 50 W in a Misonix sonicator (Misonix Inc., Farmingdale, NY). Insoluble debris was pelleted at 2000× g for 5 min, and supernatants were stored at −80°C. Samples were analyzed by SDS-PAGE as described below.