Usage
  • 47 views
  • 47 downloads

Exploiting novel Acinetobacter Oligosaccharyltransferases for the development of glycoconjugate vaccines against the Pneumococcus

  • Author / Creator
    Nasr, Mohamed A.
  • Vaccines bring many diseases under control, an example of which is the Streptococcus pneumoniae, or the Pneumococcus. Pneumococcus is one of the leading causes of meningitis in children under 5 years, and is responsible for almost a million child deaths annually, with more than 90 serotypes identified. The discovery of antigenicity of the capsular polysaccharide (CPS) is the basis for current Pneumococcus vaccines, as it is used as a serotype specific polysaccharide-only vaccine. These were reported to be effective in adults, but not in young children under 5 years. Glycoconjugate vaccines, where CPS subunits are conjugated to immunogenic proteins, revolutionized immunization against the Pneumococcus, as their efficacy was demonstrated in young children. A Pneumococcus conjugate vaccine was first introduced in the year 2000 against 7 serotypes. This vaccine has shown exceptional efficacy and is produced by chemically conjugating CPS to an immunogenic protein. This process has a lot of drawbacks including product heterogeneity and high costs. Bacterial glycosylation systems have been used as an alternative to overcome these drawbacks and produce affordable vaccines. A problem with that is the glycan and protein specificity of the conjugating enzymes, the oligosaccharyltransferases (OTases). Continuous probing for OTases with different specificities is essential for driving the glycoconjugate industry forward. In this thesis, we characterize the protein O-linked glycosylation system of members of the genus Acinetobacter. O-linked glycosylation is mediated by O-OTases, enzymes that transfer glycans from a lipid carrier to serine and threonine residues of acceptor proteins. We employ the clinical isolate A. nosocomialis M2 and the environmental isolate A. baylyi ADP1 to show that most Acinetobacter spp. encode two O-OTases with different acceptor protein specificities, contrary to previously characterized O-glycosylation systems which encode a single O-OTase. One of these OTases is specific for Type IV pilin (Tfp) glycosylation and the other one glycosylates multiple proteins. Pilin-specific OTases in Acinetobacter resemble the TfpO O-OTase from Pseudomonas aeruginosa whereas general OTases resemble the Neisseria meningitidis PglL. A. baylyi is an exception however, as both OTases are closely related to PglL with one being a general OTase and the other specific for glycosylation of the Tfp-like protein ComP. In the second part of this thesis, we attempt to exploit the A. baylyi PglLs in the glycoconjugate vaccine industry. We characterize the glycan specificity which is believed to depend on the sugar residue at the reducing end of the glycan, the first sugar added to the lipid carrier and the one in contact with serine or threonine residues of acceptor proteins. We show that both PglLs have very similar glycan specificity to the well-characterized PglL from Neisseria meningitidis. We also show that only one of the PglLs, designated PglLComP, successfully transfers Pneumococcus CPS subunits to the acceptor protein ComP. Pneumococcus CPS have a glucose residue at the reducing end and were never demonstrated to be transferred by the previously identified OTases. This is an unprecedented finding that greatly expands the tools available for glycoengineering.

  • Subjects / Keywords
  • Graduation date
    2016-06
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R3Z60C696
  • 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.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Master's
  • Department
    • Department of Biological Sciences
  • Specialization
    • Microbiology and Biotechnology
  • Supervisor / co-supervisor and their department(s)
    • Mario Feldman (Biological Sciences)
  • Examining committee members and their departments
    • Katharine Magor (Biological Sciences)
    • Tracy Raivio (Biological Sciences)
    • Christine Szymanski (Biological Sciences)