Effect of mce3R deletion on Mycobacterium tuberculosis response to acidic pH and cholesterol

The purpose of this study was to determine (i) the interplay between Mycobacterium tuberculosis response to acidic pH and cholesterol, two signals experienced concurrently by the bacterium during host colonization, and (ii) the role of the transcription factor Mce3R in regulation of this response. Overall design: Log-phase Mycobacterium tuberculosis (Mtb) strain CDC1551 (streptomycin-resistant) or its isogenic mce3R deletion mutant (OD600 ~0.6) were used to inoculate standing T25 flasks with filter caps at an OD600 = 0.3, containing 10 ml of the following media: (i) 7H9, pH 7.0, (ii) 7H9, pH 5.7, (iii) cholesterol, pH 7.0, or (iv) cholesterol, pH 5.7. After four hours of exposure to the specific environmental conditions in a humidified, 37°C, 5% CO2 incubator, Mtb samples were collected and RNA isolated. Library preparation was with the Illumina stranded total RNA with Ribo-Zero Plus kit, and barcoded samples were pooled and sequenced on an Illumina HiSeq 2500 (single-end 100 bp reads). Two biological replicates were prepared for each condition.