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Biochemical and molecular characterisation of cell wall glycosyltransferases in Mycobacterium tuberculosis

Mishra, Arun Kumar (2010)
Ph.D. thesis, University of Birmingham.

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Abstract

The human pathogen and aetiological agent of tuberculosis, Mycobacterium tuberculosis has a cell wall architecture similar to the non-pathogenic bacterium Corynebacterium glutamicum. The availability of their genome sequences has enabled the utilisation of C. glutamicum as a model for the identification and study of essential mycobacterial genes involved in the synthesis of cell wall components such as lipomannan (LM), lipoarabinomannan (LAM) and arabinogalactan (AG). In this study, we have analysed several uncharacterised open reading frames, which encode for putative glycosyltransferases from M. tuberculosis and deleted their respective orthologues in C. glutamicum. Mutant phenotypes were characterised biochemically using, two-dimensional-thin layer chromatography, SDS-Polyacrylamide-gel electrophoresis, gas-chromatograpgy-mass spectrometry, nuclear magnetic resonance spectrometry and in vitro enzyme assays. Mutants with altered phenotypes were complemeted with their respective mycobacterial orthologues to characterise their functions. In this thesis we have identified and characterised several putative glycosyltransferases and established their role in M. tuberculosis cell wall biogenesis. One of these ORFs, Rv2174/NCgl2093, was identified to encode for an α(1$$\rightarrow$$6) mannosyltransferase [MptA] involved in the later stages of the biosynthesis of the α(1$$\rightarrow$$6) mannan core of LM/LAM, while Rv1459c/NCgl1505 [MptB] was shown to be involved in the early stages of the biosynthesis of the α(1$$\rightarrow$$6) mannan core of LM/LAM. The disruption of NCgl2106 [Rv2188c] has shown its role in synthesis of phosphatidyl-myo-inositol dimannoside (Ac$$_1$$PIM$$_2$$) and also sheds further light on the synthesis of a Mannosyl-α-D-glucopyranosyluronic acid-(1$$\rightarrow$$3)-glycerol (ManGlcAGroAc$$_2$$) anchored LM (Cg-LM-B). Furthermore, three different glycosyltransferases from C. glutamicum were characterised and on the basis of biochemical analysis of mutants, NCgl2100 and NCgl2097 were identified as α(1→2) mannopyranosyltransferases [MptC and MptD], and NCgl2096 as an α(1→2) arabinofuranosyltransferase [AftE], involved in LM/LAM biosynthesis. Altogether, these studies have shed further light on the complex cell wall biosynthesis in Corynebacterineae and identified several potential new drug targets for tuberculosis.

Type of Work: Ph.D. thesis. Besra, Gurdyal Singh Colleges (2008 onwards) > College of Life & Environmental Sciences School of Biosciences QH301 Biology University of Birmingham 620
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