Deciphering mycolic acid transport in Mycobacterium tuberculosis

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description EU FP7
Amount € 500,000 (EUR)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2012 
End 11/2017
 
Description Medical Research Foundation Equipment Grant
Amount £45,000 (GBP)
Organisation Medical Research Council (MRC) 
Department Medical Research Foundation
Sector Charity/Non Profit
Country United Kingdom
Start 11/2014 
 
Description Royal Society Internatinal Exchanges
Amount £12,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 05/2014 
End 04/2016
 
Description Vacation Studentship
Amount £1,440 (GBP)
Organisation Society of General Microbiology 
Sector Charity/Non Profit
Country European Union (EU)
Start 06/2012 
End 08/2012
 
Title Conditional M. smegmatis mmpL mutant strain 
Description Conditional mutant of a mycobacterial strain higlighting the essentiality of a lipid transporter for viability (and thus its potential as a drug target) 
Type Of Material Cell line 
Provided To Others? No  
Impact In parallel with others we have for the first time identified the gene responsible for encoding the transporter for mycolic acids, key components of the mycobacterial cell wall responsioble for both virulence and cell viobility. 
 
Description Role of lipids in Mycobacterium tuberculosis-host interactions 
Organisation University of Minho
Country Portugal 
Sector Academic/University 
PI Contribution Dr.Saraiva will benefit from access to mutants from Dr.Bhatt's lab and to lipid extraction methodologies. In the future the project could expand to include animal models of infection.
Collaborator Contribution Dr.Bhatt has the opportunity to expand his projects to include TB-immunology via the specialised expertise of Dr.Saraiva.
Impact Successful application for a Royal Society International Exchanges Award.
Start Year 2013
 
Description Structural modelling of MmpL proteins from mycobacteria 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Our interests are in deciphering moecular mechanisms of lipid transport in mycobacteria, and innparticular in the MmpL gene family. This has enhanced the scope of Dr.Bavro's research-he studies the structural biology of efllux pumps and MmpLs are fro the RND family of efflux pumps.
Collaborator Contribution Generation of computer models of MmpL proteins which had helped in generating hypothesis with regards to amino acid residues that are critical for function.
Impact This collaboration in multidisciplinary.
Start Year 2013
 
Description The use of Corynebacterium glutamicum as a surrogate to study mycolic acid biosynthesis 
Organisation Julich Research Centre
Department Institute of Bio and Geosciences 1 (IBG-1)
Country Germany 
Sector Academic/University 
PI Contribution We conducted studies in parallel to identify mycolic acid transporters in mycobacteria
Collaborator Contribution Helped in construction of C. glutamicum mutants responsible for mycolate transport
Impact Resulted in a publication in the Cell Press journal Chemistry and Biology
Start Year 2012