Mycolic acid biosynthesis and processing in Mycobacterium tuberculosis

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.

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.