Uncovering the mechanisms of ABC transporter glycoconjugate translocation in Mycobacterium tuberculosis and their roles in virulence.

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Tuberculosis and leprosy are ancient infectious diseases caused by intracellular bacteria of the genus Mycobacterium. They have affected humanity for thousands of years and such long-term 'success' is closely linked to how their cell envelope, which is reminiscent of a molecular fortress, is structured and interacts with host cells. Building a molecular fortress involves complex metabolic pathways and requires exquisite coordination between multi-enzyme factories that produce the parts inside the cell before being exported outside. Understanding these export processes in detail opens new options for therapeutic intervention. The mycobacterial cell envelope differs from that of many other bacteria (pathogenic or not) in that it includes several distinct molecular layers such as peptidoglycan, polysaccharides, unusually long fatty acids named mycolic acids as well as phospholipids, collectively providing a shield against the destructive forces of antibiotics or the human immune system. Past research has largely clarified the identity of the enzymatic factories and their final products, but surprisingly little is known of how intermediate products are 'moved' across the inner membrane, before being passed to external 'assembly lines'. IN PREVIOUS WORK, we have identified two mycobacterial proteins, Rv3781 (Wzm) and Rv3783 (Wzt) as being involved in moving an essential polysaccharide called galactan across the inner cytoplasmic membrane. Rv3781 and Rv3783 are part of a much larger family of mycobacterial proteins called ATP Binding Cassette (ABC) Transporters. These membrane bound proteins require the energy (in the form of ATP) to export molecules across the cytoplasmic membrane in which they 'sit'. By sequence analysis we have identified two proteins, Rv1272c and Rv1273c, that share features similar to a well characterised ABC transporter (MsbA) in Gram negative bacteria. We show that Rv1272c plays an important role in assembly of the mycobacterial inner cytoplasmic membrane and that it is likely 'supported' by its partner protein, Rv1273c.The HYPOTHESIS leading to this proposal is that mycobacteria orchestrate glyconjugate transport by utilising specific ABC transporter proteins to 'move' these important glycan structures across the inner cytoplasmic membrane. It is important to investigate these process because they play key roles in assembly of the mycobacterial cell wall core and produce key molecules that 'hijack' the host immune response.WE PROPOSE to systematically characterise the function of ABC transporter proteins in a multi-pronged approach. First, we will generate direct and/or conditional gene knock-outs (using a genetic method known as CESTET) and investigate the effect of deleting individual ABC transporter genes on mycobacterial survival and cell envelope composition. Secondly, we will comprehensively characterise three key ABC transporter systems, that we have previously identified from preliminary work, in terms of what physiological substrates they bind and how the expression of these genes interact with the global transcriptional profile of the organism. This effort will entail comprehensive biochemical analysis of the cell envelope using specific radio-labelled tracer molecules, techniques such as mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy to determine atomic structures and RNA-seq combined with RT-PCR to establish transcriptional networks associated with ABC transporter gene expression. We will also investigate the importance of ABC transporter function of how mycobacteria are engulfed by macrophages (immune cells responsible for eradicating microorganisms). Finally, by utilising a mouse model of acute TB infection through a collaborative partnership with Dr Joanne Bacon at Public Health England, we will probe the in vivo essentiality of ABC transporters involved in immune-regulation of the host.