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Molecular and biochemical characterisation of novel glycosyltransferases in Mycobacterium tuberculosis

Birch, Helen L (2011)
Ph.D. thesis, University of Birmingham.

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Abstract

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several antitubercular drugs. Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan. The anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB and also targets the biosynthesis of the important immunomodulatory molecule lipoarabinomannan (LAM), through inhibition of Mt-EmbC. A bioinformatics approach identified putative integral membrane proteins in Mycobacterium smegmatis, M. tuberculosis and the closely related species Corynebacterium glutamicum, with features common to the GT-C superfamily of glycosyltransferases. A novel arabinofuranosyltransferase, AftC, was deleted from both M. smegmatis and C. glutamicum and shown to be an internal branching α(1→3) arabinofuranosyltransferase involved in arabinogalactan biosynthesis. Further studies revealed a truncated LAM whereby the arabinan domain was severely reduced and consisted of a simple linear arabinan of approximately 12-15 α(1→5) linked Araf residues. This mutant LAM was also shown to be a potent stimulator of TNF-α production using a human macrophage cell line, thus illustrating that masking of the mannan core by arabinan in wild type LAM alters its ability in the production of this cytokine. We also describe a further arabinofuranosyltransferase, AftB. Deletion of its orthologue in C. glutamicum resulted in a viable mutant and biochemical analysis revealed the complete absence of terminal β(1→2)-linked arabinofuranosyl residues. Further analysis confirmed AftB as a terminal β(1→2) arabinofuranosyltransferase, which was also insensitive to EMB. The bioinformatic search for cell wall glycosyltransferases led to the identification of a rhamnosyltransferase in C. glutamicum, RptA. Deletion resulted in a reduction of terminal-rhamnopyranosyl linked residues and as a result, a corresponding loss of branched 2,5-linked arabinofuranosyl residues. Furthermore, analysis of base-stable extractable lipids from C. glutamium revealed the presence of decaprenyl-monophosphorylrhamnose, a putative substrate for the cognate cell wall transferase. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and represent potential new drug targets.

Type of Work:Ph.D. thesis.
Supervisor(s):Besra, Gurdyal S.
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Additional Information:

The author has written additional papers at http://eprints.bham.ac.uk/307/ http://eprints.bham.ac.uk/118/ http://eprints.bham.ac.uk/7/

Subjects:QR Microbiology
Institution:University of Birmingham
ID Code:1597
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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