RNA-seq and genome-wide CRISPRi and Tn-seq analyses of BVL3572S-treated Mycobacterium tuberculosis H37Rv
RNA-seq and genome-wide CRISPRi and Tn-seq analyses of BVL3572S-treated Mycobacterium tuberculosis H37Rv
Cultures were harvested by centrifugation at 4,000 x g for 5 min. 3 x 1mL Mtb CRISPRi library aliquots (RLC12, Addgene 163954) were thawed and inoculated in 10 mL of 7H9 containing kanamycin 20 µg/mL (OD600 nm = 0.1). After a 5-day incubation at 37 °C, cultures were centrifuged to get rid of clumps (100 x g, 2 min) and the supernatant was diluted to an OD600 nm of 0.1 in 40 mL of 7H9 containing kanamycin and anhydrotetracycline (ATc) at 100 ng/mL. Target pre-depletion was performed for 5 days after which, medium was renewed by centrifuging the cultures (3,500 x g, 5 min) and resuspending the pellet in 7H9 before dilution to an OD600 nm of 0.05 in 20 mL of kanamycin- and ATc-containing 7H9.  Treatment was initiated by adding sub-MIC BVL3572S concentrations alone (0.85, 0.43, or 0.21 µM), with L-His 3 mM (0.43, 0.21, or 0.11 µM), with L-Ala 3 mM (1.70, 0.85, or 0.43 µM), or with 3 mM each of both L-His and L-Ala (13.63, 6.81, or 3.41 µM) to triplicate cultures. Antibiotic-free controls were prepared by adding DMSO 0.1% to cultures containing the corresponding amino acid. Cultures were outgrown for 14 days. On days 5 and 10 of outgrowth, medium was renewed by centrifuging the cultures and resuspending the pellets in medium before dilution to an OD600 nm of 0.1 in 7H9 containing kanamycin, ATc, and if applicable, BVL3572S and amino acid at the indicated concentrations. On day 14 of outgrowth, the impact of BVL3572S treatment on each culture was determined by measuring OD600 nm (the OD600 nm of untreated cultures was approximately 1). Cultures with partially drug inhibitory BVL3572S concentrations (growth reduction of 20 to 50%) and the corresponding controls were processed for genomic DNA extraction. Retained BVL3572S concentrations were 0.43 µM for the drug alone, 0.21 µM in the presence of L-His, 1.7 µM in the presence of L-Ala, and 6.81 µM in the presence of both amino acids.  Genomic DNA was isolated from bacterial pellets using the CTAB‐lysozyme method. Pellets were washed in 1 ml PBS + 0.05% Tween-80 before being resuspended in 800 μl TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and 50 µl lysozyme (15 mg/ml) and incubated at 37 °C for 16 h. 70 μl of 10% SDS and 5 μl of proteinase K (20 mg/ml) were added, and samples were incubated at 65 °C for 30 min. Next, 200 μl of NaCl 2.5 M and 80 μl of 10% CTAB were added, and samples were incubated at 65 °C for 30 min. Finally, 750 μl of ice-cold chloroform was added and samples were mixed. After centrifugation at 16,000 × g for 5 min and extraction of the aqueous phase, samples were removed from the biosafety level 3 facility. Samples were incubated with 25 μg RNase A for 30 min at 37 °C, followed by extraction with phenol:chloroform:isoamyl alcohol (pH 8.0, 25:24:1, Thermo Fisher BP1752I-400), then chloroform. Genomic DNA was precipitated from the final aqueous layer with the addition of 0.015 volume of 4 M sodium acetate and 0.6 volume of isopropanol. Mixtures were incubated for at least 2 h at -20°C. DNA pellets were centrifuged at 16,000 × g for 30 min at 4 °C and washed twice with 750 μl 80% ethanol. Pellets were dried and resuspended with nuclease-free water. DNA was quantified using the Qubit dsDNA BR assay kit (ThermoFisher Scientific). The sgRNA-encoding region was amplified from genomic DNA exactly as described in Li et al. Nature Microbiology 2022 (DOI: 10.1038/s41564-022-01130-y). Amplicons (~230 bp) were purified using NucleoMag magnetic beads (Macherey-Nagel 744970) using a double-sided size selection (bead:sample ratio of 0.6 followed by 1.0) and quantified using the Qubit dsDNA BR assay kit (ThermoFisher Scientific). Amplicon size and purity were visualized on a 1 % agarose gel and an Agilent 2100 bioanalyzer. For a more precise quantification, a qPCR assay was performed using the KAPA SYBR FAST qPCR kit (Roche KK4600). 