RNA Sequencing of intra-phagosomal Mtb populations

Heterogeneity of host cells as well as bacteria residing within them has been known to induce drug tolerance in pathogens. In Mycobacterium tuberculosis, particularly, drug tolerance within host is a major hurdle in the path to attain a sterlising cure. Reports have shown how residence of Mtb within macrophages makes the pathogen refractory to anti-TB therapy. However, the mechanisms responsible for induction of tolerance to antibiotics, particularly in resting macrophages where Mtb continues to actively replicate, is yet to be deciphered. Reports have suggested that heterogeneity induced in Mtb by cues sensed in the host environment could be a contributing factor to drug tolerance. With regard to this, differences in redox physiology of intra-macrophage Mtb (mid-point potential of the major cytosolic redox buffer in Mtb, mycothiol- EMSH) have been shown to play a role in influencing antibiotic-mediated killing. In this study, we have attempted to analyse the trascriptomic profiles of individual redox sub-populations of Mtb from within macrophages- viz., the EMSH-reduced bacteria with mid-point potential ranging between -285 and -310 mV, shown to be most refractory to killing by front-line anti-TB antibiotics inside macrophages, as well as EMSH-basal bacteria with mid-point potential ranging between -270 and -280 mV, with a much higher degree of antibiotic susceptibility within macrophages. Analysing gene expression level differences between these bacterial sub-populations would help in providing a mechanistic understanding of drug tolerance in Mtb, bred from phenotypic heterogeneity in the pathogen. The study involves profiling mRNA signatures of redox differential sub-populations of Mtb H37Rv (expressing the biosensor Mrx1-roGFP2) isolated from within PMA-differentiated THP1 monocytes at 24 hours post-infection. The profiles of EMSH-reduced and EMSH-basal intra-macrophage subpopulations were compared with in vitro grown Mtb, under similar conditions with the exception of the extracellular environment as faced within macrophages. Samples for all three conditions were prepared in duplicates. Acidic pH is one of the earliest signals faced by Mtb within the host and possibly contributes to the emergence of the EMSH-reduced sub-population during infection. In order to determine the overlap of deregulated Mtb genes between the EMSH-reduced fraction arising inside macrophages and those induced under acidic pH exposure, WT Mtb was exposed in vitro to neutral (6.6) and acidic (4.5) pH and genes induced at low pH were analyzed. Additionally, WhiB3 is a known regulator of acidic pH-mediated emergence of EMSH-reduced population of Mtb. As a control, a genetic knock-out of WhiB3 in the background of WT Mtb, along with its complemented strain, were also exposed to neutral and acidic pH. This in vitro analysis was conducted to determine the overlap of differentially expressed genes between the EMSH-reduced sub-population and the WhiB3-specific, low pH induced regulon of Mtb. Samples for all three strains at pH 6.6 and pH 4.5 were prepared in duplicates.