Molecular and biochemical characterisation of novel glycosyltransferases in Mycobacterium tuberculosis

Birch, Helen L (2011). Molecular and biochemical characterisation of novel glycosyltransferases in Mycobacterium tuberculosis. University of Birmingham. Ph.D.

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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:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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