Deciphering mycolic acid transport in Mycobacterium tuberculosis

Tuberculosis is a global health problem, further compounded due to the rise of multi-drug resistance. The causative bacterium, Mycobacterium tuberculosis has a waxy, protective cell wall which is rich in lipids. One such 'building block' of the cell wall is a group of fatty acids termed mycolic acids. While we know how mycolic acids are made, not much is known about how they are transported to the outside of the cell. This study aims to decipher mycolic acid transport processes by using an array of molecular tools that will identify genes responsible for mycolic acid transport. As mycolic acids are essential for the survival of M. tuberculosis, genes identified in this study have potential to be targeted for drug development. Additionally, as mycolic acids are also required for virulence, these studies will, indirectly, also have implications for our understanding of how M. tuberculosis causes disease.

Mycolic acids are vital components of the unique cell wall of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), and are essential for viability and virulence. While the core processes of mycolic acid biosynthesis are now well understood, not much is known about the post biosynthesis processing of newly synthesised mycolic acids inside the cell and their subsequent transport for transfer to cell wall components. The aim of this study is to identify mycolic acid transport mechanisms in M. tuberculosis and this will be achieved by:
1) Generation and characterisation of M. tuberculosis and M. smegmatis conditional mutant strains of mmpL3, a gene encoding putative large transmembrane protein, orthologs of which have been shown to play a role in mycolate transport in the related corynebacteria. A phage based Specialised Transduction method will be used to generate conditional Mycobacterium mutants of mmpL3.
2) Generation and characterisation of mutants/conditional mutants of a group of core mycobaterial genes immediately surrounding mmpL3, including a second mmpL gene, mmpL11
3) Conducting localisation studies with MmpL3 (and MmpL11)-GFP fusions using flourescence microscopy as well as Transmission Electron Microscopy.
4) Isolation of interacting partners of MmpL3 (and MmpL11) with the potential for identifying late processing enzymes that may interact with MmpL3. Bacterial two-hybrid systems, yeast two-hybrid systems and a novel nanodisc method that incorporates co-immunoprecipitation will be used in parallel to achieve this aim.

1) Direct Impact: The proposed research will have a direct impact on tuberculosis (TB) research and the immediate beneficiaries will be the PI's research group in particular, and on a wider scale the TB research community.
(i) PDRA: The PDRA employed on this project will be joining a University that hosts one of the largest molecular microbiology research clusters in the U.K. giving him/her exposure to world class research. The recent setting up of the Institute of Microbiology and Infection at the School of Biosciences will further impact the research experience of the PDRA. The PDRA will also gain training in a wide range methodologies, and coming from a different lab will also potentially contribute new ideas and expertise to the PI's existing research group. With many sections of this project at a stage where characterisation can begin immediately (eg. the existing mmpL mutants) and the high impact nature of the proposed work, a substantial output in the form of publications is expected. The School of Biosciences also has a mentoring scheme termed 'Personal Best' that ensures that the PDRA's training needs and career opportunities are identified.
(ii) The PI's group: The research group will also benefit from this work as new avenues of research are opened, expanding the themes of the laboratory. In the future this could lead to international collaborative opportunities with groups having complementary expertise.
(iii) The Research Community: Cell wall biology and particularly mycolic acid metabolism are internationally competitive areas of TB research and a huge gap exists in our knowledge of how mycolic acids are processed and transported. Thus, findings from this research will significantly move forward our understanding of how these vital lipids are exported to the cell wall of M. tuberculosis. Apart from contributing to the biochemistry and genetics of how mycolates are made, this research will also have a general impact in the areas of bacterial cell wall biosynthesis and bacterial transport mechanisms.

2) Future Beneficiaries beyond the timescale of the project:
(i) Industry: Mycolic acids are essential for Mycobacterium viability and thus genes identified from this research could potentially be developed as drug targets. Should this be the case, mechanisms in place at the University of Birmingham will be tapped into to look at potential links with industry. Prospective MRC and BBSRC CASE studentships would also be potential benefits for the PI.
(ii) Global Health and the General Public: TB is a global scourge with nearly 300,000 people getting infected every year. The severity of the disease has been compounded by the fact that multi drug-resistant (MDR) and extensively drug resistant (XDR) TB is now widespread in some parts of the world. More locally, TB has now made a comeback in the U.K. with community outbreaks being reported. As TB is a global threat, any research that furthers our understanding of the disease and the causative agent has potential impact in the fight against this scourge. In particular, this research has the potential to identify future drug targets. This is especially relevant as new anti-TB drugs are now urgently needed as the threat of MDR and non-treatable XDR TB is omnipresent. Any advance in the fight against this global disease is likely to enhance the quality of life in affected communities and individuals worldwide.