Testing of tunicamycin analogue efficacy against Mtb growing in infected macrophages
J774A.1 mouse macrophage cells were grown in J774 growth medium consisting of DMEM GlutaMAX (Gibco) supplemented with 10% fetal bovine serum, 20 mM HEPES + 0.5 mM sodium pyruvate, seeded in sterile tissue culture treated 24-well plates (Corning) at 2.5x105 cells/well and allowed to attach for 24 h in J774 growth medium. Mtb H37Rv ATCC was grown to OD650nm of 0.2 in 7H9/ADC/Tw, harvested, resuspended in J774 growth medium, filtered through a 5 μm filter to ensure a single cell suspension and diluted to 2.5x107 cells/ml. Cells were infected with 0.1 mL Mtb cell suspension at a multiplicity of infection (MOI) of 10:1 and allowed for 24 h. After infection, the medium was aspirated and the monolayer of cells washed twice with Dulbecco’s PBS and subsequently fed with 1 ml of J774 growth medium containing the compounds at the indicated concentrations in triplicate wells for each drug concentration and time point. Rifampicin was used as positive control and methanol and DMSO used for the negative controls. After 3 and 7 days of treatment, cells were lysed by 0.1% SDS, appropriate dilutions made in 7H9/ADC/Tw and plated in duplicate on solid medium consisting of Middlebrook 7H11 (Becton Dickinson) supplemented with OADC [final concentration of 0. 5% bovine serum albumin fraction V, 0.08% NaCl, 0.2% glucose, 0.2% glycerol, 0.06% oleic acid]. Colony counts were enumerated after 4 weeks of incubation.