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Permanent link (DOI): https://doi.org/10.7939/R31J97M3H

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Exploring the Glycome of Acinetobacter baumannii Open Access

Descriptions

Other title
Subject/Keyword
Glycomics
Pathogenesis
Glycosyltransferase
Carbohydrates
Glycosylation
Microbiology
Acinetobacter baumanii
Capsule
Polysaccharide
Oligosaccharide
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Lees-Miller, Robert G
Supervisor and department
Feldman, Mario (Biological Sciences)
Examining committee member and department
Glover, Mark (Biochemistry)
Szymanski, Christine (Biological Sciences)
Owttrim, George (Biological Sciences)
Department
Department of Biological Sciences
Specialization
Microbiology and Biotechnology
Date accepted
2015-01-21T08:58:03Z
Graduation date
2015-06
Degree
Master of Science
Degree level
Master's
Abstract
Acinetobacter baumannii remains a poorly understood, but very dangerous opportunistic pathogen. Though it lacks clear virulence traits expected of pathogens, it none the less poses a massive threat to the hospital system due to its inherent and acquired resistance to antibiotics, survival on abiotic surfaces, and its ability to colonise almost any part of the body. We had previously set out to identify potential virulence factors and found protein O-glycosylation, a post-translational modification attaching oligosaccharides to proteins on the bacteria's surface. To elucidate the function and synthesis of this modification, we identify here a locus in the A. baumannii genome with all the necessary genes, as well as potentially linking the process of O-linked protein glycosylation with capsular polysaccharide synthesis, large polymers of sugar on the cell surface that protect from the immune system, antibiotics, and dessication. Using microscopy, protein and carbohydrate staining, and NMR we demonstrated that A. baumannii uses the same oligosaccharide for protein glycosylation and capsular polysaccharide through mutation of a critical enzyme, PglC, which transfers the first sugar to a lipid carrier in the membrane such that it can be used in later processes. We demonstrated that mutation of PglC caused drastic reductions in biofilm formation and virulence of this dangerous pathogen through serum killing assays and murine septicaemia challenges. The essential nature of this protein led us to further investigate its characteristics, and make progress in developing an in vitro assay that could be used to screen for potential inhibitors, with the end goal of developing new antibiotics. The diversity of A. baumannii glycans, and some initial observations lead us to investigate whether PglC may be promiscuous in its substrate preferences, which would be a radical departure from existing dogma surrounding this family of proteins. Through three in vivo models we provide preliminary evidence that this is the case, and that PglC substrate specificity can be modified through single amino acid substitutions. Taken together, we provide some necessary first steps in understanding and exploiting a new enzyme for biotechnology applications and a novel target for antibiotic development.
Language
English
DOI
doi:10.7939/R31J97M3H
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
Citation for previous publication
“A Common Pathway for O-Linked Protein-Glycosylation and Capsule in Acinetobacter baumannii” Robert G. Lees-Miller, Jeremy A. Iwashkiw, Nichollas E. Scott, Andrea Seper, Evgeny Vinogradov, Stefan Schild, and Mario F. Feldman. Molecular Microbiology 12 July 2013 Issue 89(5), pages 816-830.

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