Structural and functional characterization of two Mycobacterium tuberculosis transporters

Antimicrobial resistance poses a great threat in the modern world, by rendering current treatments ineffective. Mycobacterium tuberculosis is an ancient human pathogen that is acquiring resistance to an increasing number of antibiotics, making the discovery of alternative treatments an urgent need. Bacterial efflux pumps are membrane embedded proteins that mediate active transportation of molecules across the plasma membrane. They have been implicated in the development of resistance by reducing the intracellular concentration of antibiotics, as was demonstrated by resistance to bedaquiline. This signified that the rational design of a molecule that targeted such drug transporters and inhibited their function could be a promising way of preventing or reversing antimicrobial resistance or tolerance. The M. tuberculosis genome reveals two efflux pumps, Rv3239c and Rv3728, which have not yet been structurally or functionally characterised, that could potentially enable bacterial adaptation and survival. They belong in the Major Facilitator Superfamily (MFS) of bacterial transporters. Rv3239c and Rv3728 are two of ten putative cAMP-binding proteins of M. tuberculosis. cAMP is a universal second messenger that mediates downstream cascades through interactions with proteins. cAMP binds Rv3239c and Rv3728, inducing allosterically conformational changes in the membrane embedded region that result in the transport of a molecule across the membrane. The two genes are closely related and share 61.9 % sequence identity, which could suggest comparable structures and functions.

The objective of this project is the characterization of Rv3239c and Rv3728 and their potential role in development of antibiotic resistance using a combination of structural biology, microbiology and metabolomics. The first part of the project entails production of the transporters in the non-pathogenic Mycobacterium smegmatis or another suitable expression system. Their successful expression will be followed by optimisation of a purification protocol to enable structural analysis using an appropriate technique, for example X-ray crystallography or cryo-electron microscopy. The structure, in combination with metabolomics and microbiology experiments will be used to characterize the mechanism and direction of transport and to identify the transported substrate. This is an essential step towards the potential design of drugs to interfere with or neutralize the development of resistance to antibiotics.