Identification and Exploitation of Oligosaccharyltransferase Dependent Bacterial Glycosylation Systems

• Author / Creator

  Iwashkiw, Jeremy, A

• Protein glycosylation, the covalent attachment of carbohydrates to amino acids, was once thought to be a unique attribute of Eukaryotes and select bacteria. However, both N- (attached to asparagine) and O- (attached to serine or threonine) glycosylation systems have been identified in an ever increasing number of bacteria, including various important human pathogens. Two distinct classes of glycosylation are categorized based on the presence of an oligosaccharyltransferase (OTase). For OTase-independent glycosylation, individual glycosyltransferases (GTs) transfer individual monosaccharides to proteins cytoplasmically. The other class has a more complex pathway, where individual GTs transfer monosaccharides sequentially onto a lipid carrier on the cytoplasmic face of the inner membrane, and when completed, translocated to the periplasmic face, where the OTase transfers the glycan en bloc to acceptor proteins. By exploiting the exponentially increasing number of sequenced bacterial genomes available, in silico analysis has revealed several potential OTase-dependent glycosylation systems. A homologue of Neisseria OTase (PglL) was identified in the emerging Gram negative pathogen Acinetobacter baumannii. This bacterium has been isolated from healthcare facilities for over 40 years, but was not dangerous because it was easily controllable with antibiotics. In the last decade, numerous A. baumannii isolates have developed extreme resistance to antibiotics, desiccation, and disinfectants, leading its classification as a “superbug” and one of the greatest threats to the modern healthcare system. In this thesis, a combination of genetics, molecular and cell biology, mass spectroscopy, and virulence model systems identify and characterize the general O-glycosylation system in A. baumannii. Analysis of the glycoproteome of A. baumannii revealed seven different glycoproteins modified with a pentasaccharide with a unique iii terminal subunit dependent on the OTase PglLAb. Several of these glycoproteins were recombinantly expressed and further analyzed by Western blot and mass spectroscopy. The glycosylation system of A. baumannii can be functionally reconstituted in E. coli, and PglLAb was able to transfer mono-, oligo, and polysaccharides with different structures, with relaxed glycan specificity. Bacteria require protein glycosylation for a variety of functions, including motility and pathogenesis. A. baumannii does not require O-glycosylation for swarming motility, but is essential for biofilms. The glycosylation deficient strain was non pathogenic towards Dictyostellium discoideum (macrophage model), Galleria mellonella (innate immunity response model), and was unable to compete with the wild type in colonizing and killing mice. Additionally, O-glycosylation appears to be important in infecting and killing human alveolar epithelial cells. A. baumannii was observed to cause Eukaryotic cell rounding in a glycosylation dependent manner, but did not cross link actin in vivo. It appears that O-glycosylation is highly conserved in Acinetobacter. All sequenced strains to date have a homologue of PglLAb, and a genetic locus responsible for synthesis of the O-glycan. Additionally, in this locus are the genes required for capsular polysaccharide export leading to the hypothesis a dual use for glycan. These results demonstrate A. baumannii possesses an active O-glycosylation conserved in Acinetobacter, and it is required for pathogenesis. In a related applied project, a glycoconjugate was produced by reconstituting the Campylobacter jejuni N-glycosylation system in Yersinia enterocolitica O:9. The glycosylated carrier protein was purified and the glycan was identified as the Y. enterocolitica O:9 O antigen, which is identical to the Brucella O antigen. Sera obtained iv from mice injected with the glycoconjugate was immunoreactive against both species purified LPS, but no protection was observed against Brucella infection. However, by conjugating the glycoconjugate to magnetic beads, a novel diagnostic system against brucellosis was generated.

• Subjects / Keywords

  • Brucella
  • Microbiology
  • Glycobiology
  • Diagnostics
  • Acinetobacter

• Graduation date

  Fall 2014

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3QZ22Q20

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. 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.