Tuberculosis Infection Modeling in Mice

For Mtb, mice were anesthetized and inoculated intranasally with ∼1,500 CFU of one of the Erdman strains (smyc′::mCherry, hspx′::GFP/smyc′::mCherry, and hsp60′::GFP) resuspended in 30 μl of PBS containing 0.05% Tween 80. Inoculum dosage was confirmed by plating different dilutions of the bacterial stocks in 7H10 agar plates supplemented with OADC Enrichment, glycerol, and hygromycin B. Plates were incubated at 37°C and colonies enumerated 3 wk after. 3 wk post-infection (w.p.i.), mice were sacrificed, and the lungs were aseptically removed and placed in PBS containing 5% FBS. To minimize unwanted changes in the gene expression profile of both host and bacteria, samples were kept on ice and immediately processed using a GentleMACS tissue dissociator (Miltenyi Biotec; Pisu et al., 2020b (link)). The dissociated lung material was then passed through a 70-µM cell strainer, and red blood cells were lysed with ammonium-chloride-potassium lysis buffer (Lonza).
For BCG, mice were intravenously infected with 10⁶ BCG (Pasteur) bacilli. Lung cell suspensions were obtained as detailed above.