Mycolic acid biosynthesis and processing in Mycobacterium tuberculosis

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

Abstract

The tuberculosis-causing, human pathogen Mycobacterium tuberculosis (MTB) contains a lipid rich ‘waxy‘ cell wall that forms a protective barrier against harsh environments. Mycobacterial lipids not only offer a protective cover, but are also involved in interactions with infected host cells. Furthermore, some lipid components are essential for the survival of MTB and thus the enzymes that catalyze their biosynthesis represent potential targets for antimycobacterial drugs. The proposed project aims to further our understanding about how these lipid building blocks are made by generating defined mutants of MTB in candidate genes. The mutant strains will then be tested for loss of lipid components and/or accumulation of intermediate compounds. The study also aims to assess the role of specific lipids in virulence by testing these mutant strains in laboratory models of infection. The hope is that these studies will aid in our search not only for new drugs effective against tuberculosis, but also a better vaccine.

Technical Summary

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, produces long chain alpha-alkyl, beta-hydroxy fatty acids, termed mycolic acids, that are important constutuents of its distinct, lipid rich cell envelope. Mycolic acids, synthesized by a fatty acid synthase II (FASII) complex, are found either esterified to the peptidoglycan-linked arabinogalactan complex, or present as a part of the interspersed glycolipid, trehalose dimycolate (TDM). The broad aim of the proposed project is to use null mutants, generated by allelic exchange, to help understand mycolic acid biosynthesis and processing pathways in mycobacteria, and outline their role in MTB pathogenesis. More specifically the objectives of the proposed study are:
1) Identify the genes encoding enzymes involved in a key desaturation step in mycolic acid synthesis.
2) Study the effects of mycolic acid chain length shortening in a MTB kasB deletion mutant on the virulence and immunomodulatory properties of MTB.
3) Delineate processes involved in post FASII intracellular processing of mycolic acids and their transport outside the cell.
4) Assess the importance of the above mentioned biosynthetic steps in pathogenesis by checking mutant or conditional mutant strains for surivival in macrophages and immunocompetent mice, and by purifying intermediates from these strains and testing their immunomodulatory properties in macrophages.

This study is an exercise in functional genomics, making use of information gained from the genome sequence of MTB to study mycolic acid metabolism. I plan to take a multidisclipinary approach encompassing bacterial genetics, biochemistry, chemical analysis and immunology to fulfil the objectives stated above. First, a highly efficient allelic exchange method termed Specialized Transduction will be used to generate deletion mutants in MTB, M. bovis BCG and the fast growing M. smegmatis. Next, the mutants will be thoroughly characterised using analytical techniques including TLC and HPLC. Accumulated intermediates will be purified and structural analysis will be done using Mass Spectropscopy and NMR. Finally, to assess the role of individual genes in pathogenesis, mutant strains will be tested for surivival in mice follwing aerosol infection. In parallel, purified mycolates and TDM from mutant strains will be tested for immunomodulatory properties in macrophages using microarrays and ELISA.

Publications

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Bhatt A (2007) Deletion of kasB in Mycobacterium tuberculosis causes loss of acid-fastness and subclinical latent tuberculosis in immunocompetent mice. in Proceedings of the National Academy of Sciences of the United States of America

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Bhowruth V (2008) Tuberculosis: a balanced diet of lipids and carbohydrates. in Biochemical Society transactions

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Birch HL (2010) A truncated lipoglycan from mycobacteria with altered immunological properties. in Proceedings of the National Academy of Sciences of the United States of America

 
Description MRC New Investigator Grant
Amount £452,605 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 08/2016
 
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 Program Grant (Co-investigator)
Amount £1,455,452 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2013 
End 12/2017
 
Description Project Grant (Co-applicant)
Amount £495,902 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2010 
End 06/2013
 
Description Project Grant (Co-applicant)
Amount £487,606 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2010 
End 08/2013
 
Description Royal Society Internatinal Exchanges
Amount £12,000 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2014 
End 04/2016
 
Description UK-India Education and Research Initiative (UKIERI)
Amount £40,000 (GBP)
Organisation British Council 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2012 
End 03/2014
 
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. 
 
Title Mycolate-less strains of mycobacteria 
Description We have recently generated a mutant Mycobacterium smegmatis that is unable to make mature mycolic acids, key components of the cell wall of mycobacteria like Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). The mutant accumulates precursors instead and is more sensitive to antibiotics. We are currently extending these studies to M. tuberculosis where we expect to see an effect on virulence 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2010 
Provided To Others? Yes  
Impact Generation of an attenuated M. tuberculosis strain with potential use in the development for a vaccine for TB. The affected gene product may also be developed as a secondary drug target. 
 
Description A mouse-injection model to study effects of purified mycobacterial lipids 
Organisation Osaka City University
Department Graduate School of Medicine
Country Japan 
Sector Academic/University 
PI Contribution We have supplied mycobacterial glycolipids from wild type and mutant strains to be tested in the mouse-injection model
Collaborator Contribution This collaboration with Dr.Nagatoshi Fujiwara has allowed us to assess the immunomodulatory potential purified mycobacterial lipids, specifically trehalose dimycolate, in a murine-injection model.
Impact This collaborative work will form part of data that is currently being written up for publication.
Start Year 2008
 
Description Effect of phosphorylation on activities of mycolic acid synthesising enzymes 
Organisation National Institute of Immunology NII
Country India 
Sector Public 
PI Contribution Provide access to mutant strains defective in mycolate biosynthesis, to test hypothesis regarding the potential role of phosphorylation in regulating mycolate biosynthesis.
Collaborator Contribution Dr.V. Nandicoori is an expert on protein phosphorylation and as part of an ongoing collaboration evaluates the effect of phosphorylation on activities of mycolate biosynthesis candidates identified in our studies.
Impact Recent joint publication in the Journal of Biochemistry (PMID 20864541)
Start Year 2009
 
Description Lipids as a biomarker for TB 
Organisation Indian Institute of Science, Bangalore
Department Division of Biological Sciences
Country India 
Sector Academic/University 
PI Contribution Training of staff and PhD students from IISc bangalore in the extraction of mycobatcerial lipids. Additionally, provision of mutant strains as controls of the development of lipids as a bio marker for detecting TB bacilli using infrared spectroscopy.
Collaborator Contribution Expertise in Raman and infrared spectroscopy as methods for detecting lipids from mycobacteria
Impact Multidisclipinary collaboration which is at the halfway stage and thus no immediate outputs to report at this stage. The project is funded by the UK-India Education and Research Initiative.
Start Year 2013
 
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 Zebrafish embryo model of mycobacterial infection 
Organisation VU University Medical Center
Department Department of Medical Microbiology and Infection Control
Country Netherlands 
Sector Academic/University 
PI Contribution Generated Mycobacterium marinum strains defective in cell wall lipids
Collaborator Contribution Dr.A. van derSaar is an expert in zebrafish models of bacterial infection and tests mutants of Mycobacterium marinum generated in my group for attenuation in the zebrafish embryo model of infection.
Impact Two manuscripts which contain data that has resulted from this collaboration are currently being written up.
Start Year 2009
 
Description cAMP metabolism and its link to the mycobacterial cell wall 
Organisation Indian Institute of Science, Bangalore
Department Department of Molecular Reproduction, Development and Genetics (MRDG)
Country India 
Sector Academic/University 
PI Contribution Initiated a new collaboration with Prof.Sandhya Visweswariah, IISc Banglaore, Inida in the area of linking central metabolism to the biochemistry of the cell wall. My research team contributed to the generation of knkout strains and the biochemical analysis of mutant strains.
Collaborator Contribution Access to mutant strains.
Impact Successful funding for this pcollaboration from the UK-India Education and Research Initiative for a Thematic Partnership: Novel Approaches to Tackling Tuberculosis.
Start Year 2011
 
Description British Science Festival 2010 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I organised an 'open access' one day symposium titled 'Tackling Tuberculosis: From the laboratory to your GP', as part of the British Science Festival held in Sept 2010 in Birmingham.

The symposium provided a common forum for scientists, clinicians and industry to present their work and discuss approaches to be taken to fight the global menace of TB.
Year(s) Of Engagement Activity 2010