Targeting Trehalose and Methylglucose Lipopolysaccharide Biosynthetic Pathways in M. tuberculosis - Structural and Functional Characterisation, and Early-Stage Drug Discovery of OtsA and Rv3030

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is one of the most successful pathogens, with much of its virulence predominantly associated with its thick, lipid rich cellular envelope that modulates the host immune response. Tuberculosis, having a long history, has proved to be a challenging disease to eradicate. In mycobacteria, trehalose occurs as a free sugar in the cytoplasm, as well as a constituent of cellwall glycolipids, such as cord-factor, sulfolipid-1 and lipooligosaccharides. M. tuberculosis possesses two pathways for synthesis of trehalose, the OtsA-OtsB pathway and the TreY-TreZ pathway. Multiple biosynthetic pathways underline the critical role that trehalose plays in the bacteria. OtsA-OtsB is the dominant pathway, required for bacterial growth in laboratory culture and for virulence in a mouse model. OtsA, a retaining glucosyltransferase encoded by the otsA (Rv3490) gene, catalyzes the synthesis of trehalose-6-phosphate from ADP-glucose and glucose- 6-phosphate, which is subsequently dephosphorylated by a functional homologue of OtsB (encoded by otsB2/Rv3372 gene) to yield trehalose. Both otsA and otsB2 gene have been shown to be essential for the growth of M. tuberculosis. The present investigation focuses on this pathway, more specifically on OtsA. The recombinant orthologous protein from M. thermoresistibile was structurally and functionally characterised. The crystal structure revealed that there are two domains, with the catalytic site at their interface. The biochemical and structural data indicated that the enzyme had high preference for ADP-glucose as a glucose-donor. The protein activity was also shown to be regulated by feedback inhibition. Furthermore, a structure-guided, fragment-based drug-discovery exercise was carried out against the target, which led to identification of fragment hits that have an inhibitory effect on the activity of the enzyme. Polymethylated polysaccharides (PMPSs) are complex intracellular polysaccharides, which are exclusive to mycobacteria and closely related species. PMPSs include methylglucose lipopolysaccharides (MGLPs) and methylmannose polysaccharides (MMPs). MGLPs, which are composed of acylated glucose and 6-O-methylglucose units, are found in all mycobacterial species, including the slow growing pathogenic M. tuberculosis. On the other hand, MMPs are linear polysaccharides that are not found in M. tuberculosis, but are found in non-pathogenic M. smegmatis and other fast growing mycobacteria. Three gene clusters (Rv1208-Rv1212c, Rv2418c-Rv2419c and Rv3030-Rv3037c), initially thought to coordinate MGLPs biosynthesis, contain several genes that are considered to be essential for M. tuberculosis growth. One of the genes Rv3030, encoding for a SAM-dependent 6-O-methyltransferase, is essential for survival of the bacilli and plays a pivotal role in the biosynthesis of MGLPs. In the present study, an orthologue of Rv3030 from M. smegmatis has been structurally characterized. To overcome a myriad of issues with crystallization, low resolution and lack of reproducibility of the crystals, an alternative approach was taken to determine the structural features of the protein by NMR spectroscopy using 15N, 13C labelled protein. The protein has the β-sheet topology of the classic Rossmann fold, and exists as a monomer in solution. OtsA and Rv3030, essential for survival of M. tuberculosis, represent attractive anti-tuberculosis drug targets, and this study focuses on understanding structural, biophysical and biochemical properties of these targets. This knowledge may then be used to identify lead chemical entities that bind to these proteins and modulate their function, providing new chemical tools that may be of use in designing antimicrobials for the fight against tuberculosis.