*6.1. Target Identification*

Mycobacteria have unique cell envelopes, high in lipid diversity and abundance, comprising up to 40% of the bacillus dry weight [100]. Cell wall biosynthetic pathways are the target of many existing anti-TB drugs; in addition, the sequencing of the *M.tb* genome revealed many lipid biosynthesis and polyketide synthase genes that might be exploited as potential therapeutic targets. The study of this network of cellular lipids within a biological system is broadly categorized in a branch of metabolomics, known as lipidomics, which examines lipid species that are present and how they interact with other lipids, metabolites and proteins in a cell [101]. Lipidomics relies on mass spectroscopy, measuring the mass-to-charge ratio and abundance of gas-phase ions, further characterized into gas chromatography (GC)-MS, liquid chromatography (LC)-MS and direct infusion-MS [102]. The large diversity of lipids and the lack of spatial information about the distribution of these moieties within a cell complicates the inferences from lipidomic experiments [103]. Lipidomics has been employed to discover potentially druggable lipid biosynthesis pathways based on the *M.tb* response to the changing environment. Raghunandanan et al. determined the pattern of *M.tb* lipid changes in hypoxia-induced dormancy and resuscitation, showing that lipid concentration drastically decreased during dormancy and gradually increased again during re-aeration [104]. Several lipids were more abundant in non-replicating bacteria, revealing potentially targetable pathways [104]. This study demonstrates the potential of lipidomics to evaluate *M.tb* in vitro in conditions predicted to mimic in vivo microenvironments. Compared to conventional high performance-LC, the ultra-performance LC technique provided a much

greater chromatographic resolution and subsequently faster analysis time [105]. Lipidomics is also a valuable tool to characterize lipid biosynthetic pathway targets. The fatty acid synthase FAS-II multi-enzyme system is essential for the biosynthesis of mycolic acids and normal cell wall function in mycobacteria. It is the target of several antimycobacterial drugs, such as isoniazid, and therefore offers additional therapeutic potential. To understand the role of HadD, a novel FAS-II enzyme, Lefebvre et al. analyzed total extractable lipids from *M. smegmatis hadD* knockout mutants by high-performance thin-layer chromatography to demonstrate that *hadD* deletion resulted in the absence of α- and epoxy-mycolic acids that disrupted the cell envelope and reduced bacterial fitness [106]. Subsequently, the authors showed that *hadD* deletion in *M.tb* resulted in a 63% reduction in keto-mycolic acids, while overexpression of *hadD* induced an 87% increase in keto-mycolic acids compared to wild type. Knockout mutants of *hadD* were attenuated in a murine model of infection, confirming *hadD* as a new target for drug discovery [107].
