Date of Award

Fall 2012

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry

Supervisor

Gerard Wright

Co-Supervisor

Marie Elliot

Language

English

Committee Member

Brian Coombes

Abstract

The glycopeptide antibiotic vancomycin is used as front line treatment for serious Gram-positive infections and resistance to this drug is widespread. Three genes are essential for resistance, vanHAX, which are controlled by a two-component regulatory system VanR and VanS. Here, glycopeptide resistance is found to be ancient and diverse in the environment. A vanA open reading frame from 30 000 yr old DNA was identified and the enzyme was shown to be as functional as comparable to modern day VanA homologs. In the environment resistance is found to be diverse and widespread. For example, the organism Desulfitobacterium hafniense Y51 VanH was shown as non-essential in conferring inducible resistance. Furthermore in the glycopeptide producer Amycolatopsis balhimycina harboring the classic vanHAX, a functional VanA homolog is described as an orphan gene outside of any recognizable gene cassette .

Glycopeptides are natural products made by members of the Actinomycete family and are modified by different types of tailoring enzymes. Of particular interest is the glycopeptide A47934, which is ‘aglyco’, and sulfated. The sulfotransferase StaL will transfer not only a sulfate group to A47934, but a sulfamide and fluorosulfonate group. Focusing on additional tailoring enzymes, the biosynthetic cluster of the sulfated glycopeptide UK68597 was sequenced. This cluster has provided a resource for glycopeptide tailoring enzymes for use to modify the A47934 backbone. Sulfation was the first focus and the substrate promiscuity of StaL was explored to expand the chemical diversity A47934 and vancomycin. This work has led to the discovery that glycopeptide sulfation will antagonize the activation and expression of vanHAX. A new sulfated vancomycin derivative was created with this antagonizing activity in the clinical pathogen Enterococcus faecium of the VanB phenotype. Implications of these results and the further use of tailoring enzymes to modify glycopeptides to antagonize resistance will be discussed.

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