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

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.

Mycobacterium tuberculosis (Mtb) has a unique cell envelope that contributes to its unusual low permeability, resistance to common antibiotics and immunomodulation of the host. This highly complex cell envelope contains a striking variety of glycoconjugate structures each with key functional roles: arabinogalactan (AG), an essential element of the cell wall core; the lipoglycan family consisting of phosphatidylinositolmannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM), subversion of the host immune response; and alpha-glucan, further immunomodulation and capsule formation. Precisely how these complex glycan structures are transported across the mycobacterial cytoplasmic membrane remains elusive. The Mtb genome encodes a ~40-strong family of ABC transporter proteins, of which only a handful have been partially investigated as drug efflux pumps or sugar importers. Based on our data, we have identified three ABC transporters with proposed functional roles for export: Rv3781/Rv3783, AG; Rv1272c/Rv1273c, lipoglycan; and Rv1217c and Rv1218c, alpha-glucan. This project will establish essentiality of these ABC transporter proteins and assign functionality for glycoconjugate translocation events that are central to mycobacterial cell envelope biosynthesis. We will investigate these proteins by probing the effect of direct and/or conditional knock-outs on cell envelope composition. Using RNA-seq, we will explore the transcriptional networks associated with ABC transporter mediated glycoconjugate movement across the mycobacterial cytoplasmic membrane. Finally, using both a macrophage and a mouse model of TB infection, we will probe the essentiality of selected ABC transporters in Mtb and their roles in virulence. The project outlined above will greatly assist our understanding of how this deadly pathogen forms its complex cell envelope, clarify the functional roles of ABC transporter proteins and provide new possibilities for the development of novel therapies.

GENERAL BIOLOGICAL AND CLINICAL CONTEXT
TB remains a major global health problem, reflected by the inclusion of TB among the WHO's Millennium Development Goals (target 6.c - "reverse the spread of the disease [tuberculosis] by 2015").
There is a broad consensus that overcoming drug-resistant forms of TB will require, among other factors, to hit novel targets in the disease-causing organism. The research in this proposal has three novel targets at its starting point, and in exploring essentiality of glycoconjugate ABC transporter systems, has potential to expand this set. While the proposal focuses firmly on biological questions and will not pursue any screening or drug discovery efforts, its outcomes will inform screening efforts elsewhere, including complementary research activity at Birmingham. We note for instance that the applicants are involved in the EU-funded CooperaTB (Marie Curie ITN, GB and LA PI and co-PI, respectively), a hit-to-lead programme that involves GSK as industrial partner.

DISSEMINATION
We will maximise dissemination of this research by publishing in Open Access journals, and presenting on both international (GRC, Keystone) and national Meetings (Microbiology Society Meetings, Acid Fast Club).

ENGAGEMENT WITH INDUSTRIAL PARTNERS
The applicants are engaged with industry, notably with GSK in the Marie Curie-funded CooperaTB project. We will explore with these partners the potential to exploit novel targets emerging from the systematic analysis of glycoconjugate ABC transporter systems, which could, for instance, lead to hit-enrichment screens, biasing the whole cell screen towards a chosen by target overexpression.

TRAINING AND CAREER DEVELOPMENT OF PDRA
The project provides a first-class training opportunity to prime an early career researcher for a career in molecular microbiology and allied areas. The project will provide the PDRA with comprehensive training in contemporary research methods, including genetic tools (e.g. CESTET), analytical methods (e.g. mass spectrometry, NMR and radiolabelling studies) and in doing experiments under Biosafety Level-3 lab conditions. Working in the dynamic environment of the Institute of Microbiology and Infection (IMI), the PDRA will participate in seminar and networking events, and thus gain a comprehensive view of modern microbiology. In addition, the PDRA will benefit from the PERCAT (Postdoctoral / Early Researcher Career Development And Training) programme. Based on this experience the PDRA will be enabled to make lasting contributions to the knowledge base of UK plc and have a successful career in academia or industry.

ENGAGEMENT WITH GENERAL AUDIENCES
All applicants have been actively engaged in outreach activities for a number of years, interacting with the general public and school children. This includes talks and presentations in local secondary schools, presentations on campus for the general public. Alderwick has authored promotional material for the University as well as presenting to and engaging with audiences on University Open Days. Alderwick has appeared on local radio stations to talk about microbiology, antimicrobial resistance and related topics, as well as visiting local schools. Such activities are increasingly part of academic activity and will continue throughout the duration of the funding period.

COMMERCIAL EXPLOITATION OF OUTPUTS
We do not expect this proposal to result in commercial products at the end of the period of support. However, should the project lead to patentable IP, we will engage with the University of Birmingham's Research and Innovation Services and its commercial arm Alta Innovations Ltd.