*4.2. Mode of Action*

Proteomics has helped to deconvolute *M.tb* responses to drug exposure providing insight into mechanisms of drug action and resistance [86]. Recently, Meneguello et al. used proteomics to explore the metabolic pathways that contribute to the activity of rifampicin [87]. A small percentage of rifampicin resistance occurs through mechanisms other than the well-characterized target, β subunit of RNA polymerase. Proteomic profiling of rifampicin-treated *M.tb* resulted in the under-expression of four proteins implicated in cell wall biosynthesis (Ino1, FabD, EsxK and PPE60), suggesting that rifampicin also affects cell wall synthesis contributing to bacterial death. The authors used a liquid chromatography-mass spectrometry (LC-MS) approach, assessing small changes to the *M.tb* proteome temporally. Consideration should be taken when conducting such experiments to minimize the introduction of false-positives as a result of weak signals from an analytical column coupled with a highly sensitive detection platform. Despite these limitations, the authors reported a protein coverage comparable to other studies that applied nano-liquid chromatography. Nano-liquid chromatography is becoming an established tool for advanced peptide separation that uses very narrow columns that are more effective for detecting low abundance compounds [88]. Similarly, proteomics has been utilized to discern the mode of action of repurposed drugs that inhibit *M.tb* in vitro. Sulfamethoxazole, a broad-spectrum antibiotic that primarily targets the folate biosynthesis pathway, exhibits a synergistic effect when combined with other anti-TB drugs. Sarkar et al. mapped the *M.tb* response to sulfamethoxazole exposure using proteomics, identifying induction of oxidative stress and electron transport chain pathways that suggested an additional mode of action for this drug [89]. Proteomics may also be exploited to determine mechanisms of drug resistance. Putim et al. employed a shotgun proteomics system to identify proteins secreted in isoniazid- and rifampicin-resistant *M.tb* compared to drug-sensitive *M.tb*. Bacterial cultures were filtered through low binding protein-cellulose acetate membranes to collect culture filtrate proteins, before sodium dodecyl sulphate-polyacrylamide

gel electrophoresis (SDS-PAGE), in-gel digestion and LC-MS. Depending on the aim of the study, consideration in the liquid chromatography (LC) approach should be taken. For example, for high proteome coverage, or for detecting proteins in low abundance, a 1D gradient for 8 h or a 2D LC should be used. However, for analysis of a limited sample volume, or where characterization of low-abundance proteins is not needed, a 1D gradient for 4 h may suffice. In addition, reproducibility and sample requirements will vary greatly depending on the method used [90]. Differential abundance of proteins involved in lipid metabolism, proteasome function and ATP-binding cassette transporters (ABC transporters) between drug-resistant and drug-sensitive strains may reveal novel systems that influence drug efficacy [91].

## **5. Metabolomics**
