## 3 Transcriptomics
### Mode of Action  
The transcriptional response of _M.tb_ to antimicrobial drug exposure has improved the understanding of many drugs, providing new insights for antibiotics currently in use for TB treatment with known cidal mechanisms, as well as predicting mode of action and the targets of novel compounds. This unsupervised approach is especially useful for understanding the actions of lead compounds from high-throughput screens where the mechanism of _M.tb_ killing is entirely mysterious. Comparison of the _M.tb_ transcriptional response to a novel compound with mRNA signatures derived from drugs of known function allows broad mode of action to be revealed (Figure 2). In the first study of _M.tb_ transcriptional adaptations to drug treatment, [PERSON] et al. used DNA microarrays to explore changes in gene expression in response to isoniazid [61]. The authors showed that drug exposure induced several genes relevant to isoniazid's known mode of action; drug treatment caused the cluster of five genes encoding type II fatty acid synthase enzymes (_fabD-acpM-kasA-kasB-accD6_) to be upregulated. Other induced genes, such as _efpA_ and _alpC_ not in the biosynthetic pathway targeted by isoniazid, were linked to the toxic effects of the drug. In subsequent years, several studies have used DNA microarrays to correlate the mRNA signatures of _M.tb_ exposed to antimicrobial drugs with predicted mode of action [62; 63; 64]. [PERSON] et al. generated a dataset of 430 _M.tb_ gene expression profiles to measure the effect of 75 different drugs, drug combinations and growth conditions [64]. The individual RNA drug signatures were classified into groups of agents with similar modes of action (protein synthesis inhibitors, transcriptional inhibitors, cell wall synthesis inhibitors and DNA damaging agents), which have been used to predict the mechanism of action of antimycobacterial compounds of unknown function derived from whole cell screening approaches [65; 66; 67]. Figure 2: The application of transcriptomics to define drug mode of action. Principle Component Analysis (PCA) of _M.tb_ responses to seven different drugs (represented by shapes) derived from RNAseq of 2–3 biological replicates per drug. _M.tb_ mRNA signatures from antimicrobial drugs with similar mechanisms of action will cluster together. Transcriptomics has been applied to investigate the mode of action of drugs now in phase I/II studies or in the clinic [2]. The _M.tb_ transcriptional signature to benzothiazinone exposure most resembled that of cell wall inhibitors and ethambuttol in particular, providing an early indication that the compound was targeting cell wall biosynthesis and arabin galactan synthesis. This was confirmed to be the case with benzothiazinones inhibiting decaprenyl-phosphoribose-2\({}^{\prime}\)-epimerase (DprE1) in the metabolism of D-arabinose, two steps upstream of the action of ethambuttol [7]. RNA profiling provided fundamental insights to elucidate the molecular mechanism of mycobacterial killing by pretomaniid (formerly PA-824), a nitroimidazole active against both replicating and non-replicating _M.tb_, by inhibiting cell wall synthesis and releasing nitric oxide [69; 70]. Pretomani exposure resulted in a dual signature indicating cell wall inhibition (similar to isoniazid) and respiratory chain disruption (similar to respiratory inhibitors such as cyanide). While the upregulation of genes such as _fasI_, the _fasII_ operon, _eprA_ and _iniBAC_ signposted the aerobic killing mechanism targeting cell wall biosynthesis, drug efficacy in anaerobic conditions was marked by the induction of the _cytd_ operon encoding the non-proton-pumping cytochrome bd oxidase, the nitrate reductase _narGHIJ_ and other genes involved in respiration [71]. RNAseq analysis of the _M.tb_ response to the recently approved nitroimidazole delamamid (formerly OPC-67683) showed many similarities to the pretomanid signature, highlighting that respiratory poisoning plays an important role in the bactericidal effect of these compounds in anaerobic conditions [72].