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NUS Graduate School for Integrative Sciences and Engineering Research Project Write-up Title of Project : Mycolic acid export to the outer membrane of mycobacteria Name of Supervisor : Dr Chng Shu Sin Contact Details: [email protected] Short Description Our research program lies at the interface of chemistry and biology, and involves the use of chemical, biochemical, genetic and biophysical approaches to characterize both the chemistry and biology of a given system. The problem that my group is interested in studying is membrane biogenesis, i.e. how a biological membrane is assembled. Membrane lipid bilayers form the basis for life, physically defining cells and organelles, and modulating the chemical environments within these compartments for optimal metabolism and growth. Despite these fundamental roles, however, our understanding of membrane biogenesis has remained rudimentary; we do not know how a cell makes more of itself. Conceptually, the assembly of a membrane involves acquisition of its protein and lipid constituents from another compartment where these molecules are synthesized. For lipids, this entails transport from one membrane to another, usually across an aqueous environment. Our research group is focused on using bacterial outer membranes as models to understand membrane biogenesis. Specifically, we are interested in elucidating the mechanisms of inter-membrane lipid trafficking in Gram-negative bacteria and mycobacteria. The cell envelope of mycobacteria consists of two lipid bilayers: an inner (plasma) membrane (IM) that encloses the aqueous cytoplasm and an outer membrane (OM) that faces the extracellular milieu. Between these two membranes is a second aqueous compartment known as the periplasm, which contains the cell wall that determines the shape of the bacterial cell. This unique double-membrane envelope renders mycobacteria resistant to external insults. The mycobacterial OM is a unique lipid bilayer whose detailed structure and composition is not that well understood. The major component of the OM is mycolic acids, which are C60-C90 long branched chain lipid moieties that covalently tether the OM to the mycobacterial cell wall. These hydrophobic mycolic acids are believed to form the entire inner leaflet of the mycobacterial OM, contributing to the low fluidity of the lipid bilayer and thus rendering the OM an effective permeability barrier. Mycolic acids are bio-synthesized as trehalose monomycolates (TMMs) in the IM, which are then transported across the IM, periplasm and to the OM where they become functionalized onto the cell wall. The biosynthesis of mycolic acids is quite well characterized. However, the final steps of TMM synthesis and transport across the cell envelope remain unknown. Mycobacterial infections, typified by tuberculosis (TB), are difficult to treat due to a variety of reasons. One of these reasons is that many classes of antibiotics (macrolides, glycopeptides, etc) are not effective because they cannot penetrate the OM. Furthermore, resistance to effective drugs (isoniazid, rifampicin, etc) is already on the rise, underscoring the need to invent new strategies to fight mycobacterial pathogens. Since the OM is essential for the survival of these pathogens, and compromising OM integrity enables the use of many antibiotics currently only effective against other pathogens that do not possess an OM, the molecular machines that build or maintain the OM represent great targets for antibiotic discovery. In particular, the biosynthetic pathway of mycolic acid, the major component of the mycobacterial OM, is a well-validated target for first and second line TB drugs. In this project, we focus our efforts on identifying proteins involved in TMM biosynthesis and transport in Mycobacterium smegmatis and characterizing known systems involved in these processes. Students in this project will be trained in areas such as basic molecular biology and bacterial genetics, phenotypic assays, membrane fractionation, lipid extraction/analysis, membrane protein expression/purification and protein-ligand interaction analysis.