Complex pathways in mycobacterial cell wall biosynthesis
thesisposted on 03.03.2017 by Cashmore, Tamaryn Jane
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Mycobacterium tuberculosis is a significant human pathogen, responsible for approximately 1.3 million deaths annually. The increase of multi-drug resistant (MDR) and extensively drug resistant (XDR) tuberculosis is of particular concern as current first and even second line medications are less or no longer effective. Current antibacterial drugs, such as isoniazid and ethambutol, target various biosynthetic pathways within the complex cell wall of these bacteria, which is a key virulence factor. Its successful defence against the host immune system is due to a largely impermeable cell wall consisting of a multi-laminate mycolyl-arabinogalactan-peptidoglycan complex (mAGP) and a glycolipid layer embedded into the cell wall. Phosphatidylinositol mannosides (PIMs), lipomannan (LM) and lipoarabinomannan (LAM), all virulence factors, are included in this glycolipid layer. Previous studies in our group identified a highly conserved genetic locus involved in mycobacterial cell wall synthesis. Gene knockout studies in Corynebacterium glutamicum, a related model species tolerant of cell wall defects that kill mycobacteria, identified three proteins involved in the transport of mycolic acid intermediates across the cell wall. We have shown that the orthologs NCgl2764, NCgl2762 and NCgl2759 are involved in mycolic acid synthesis. The aim of this study was to determine the roles of NCgl2760 and NCgl2761, the two remaining genes within the putative complex. NCgl2761 was disrupted in C. glutamicum by homologous recombination and next the mutant was studied for cell wall defects. Total lipids, PIMs LM, LAM and mycolic acids were investigated by high performance thin layer chromatography (HPTLC), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gas chromatography-mass spectrometry (GC-MS). Delayed growth and a block in the path from acetyl trehalose monocorynomycolate (AcTMCM) to trehalose dicorynomycolate (TDCM) indicate their involvement in mycolate metabolism. These conclusions were congruent with those of the other three genes in the putative complex. RNA extraction and co-transcription with NCgl2760 were additionally investigated. The results do indeed show co-transcription of C. glutamicum NCgl2761 and C. glutamicum NCgl2760. This co-transcription was replicated in Mycobacterium smegmatis, between genes MSMEG_0315 and MSMEG_0317, the orthologs of NCgl2761 and NCgl2760. Deletion mutants of NCgl2760 using the same principles were completed. Initial growth experimentation showed no delayed growth, unlike the other four genes, so the following procedures were performed in triplicate. Complementation of ΔNCgl2760 was unsuccessful, consequently slice overlap extension (SOE) was attempted. This too failed. Nevertheless, experimental procedures continued and next showed absence of a block in the mycolic acid biosynthetic pathway. But, a Pierce™ silver stain showed a truncated, faster migrating LAM of a size measuring approximately 10kDa on a 15% SDS-PAGE gel. Both these findings are novel and suggest that a complex of at least four proteins mediates mycolic acid transport, and RNA studies raise the possibility of a link between the mycolic acid and LM/LAM biosynthesis pathways in mycobacteria and corynebacteria. All these studies contribute to our understanding of mycobacterial cell wall pathways which may offer attractive drug targets.