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Exploration of novel factors associated with Group B Streptococcus virulence

  • Author / Creator
    Hulbah, Maram
  • Group B Streptococci (GBS) is the leading cause of invasive diseases in neonates, pregnant women and non-pregnant adult. In order to cause invasive disease, GBS encodes several virulence factors that are involved in the disease process. One such factor is the moonlighting glycolytic enzyme, phosphoglycerate kinase (PGK), a surface-expressed protein whose mechanism of becoming expressed on the GBS surface has not been known. The surface localization of PGK appears to contribute to bacterial virulence through its ability to bind strongly to host plasminogen. Determining the role of surface-expressed GBS-PGK through deletion of the gene for PGK is hampered by PGK’s central role in glycolysis. Consequently, it was necessary to first identify the binding ligand of PGK on the GBS surface, then prevent the expression of PGK on the GBS surface. My results identified two genes in GBS, eveA and eveB, external virulence effector A and B, that are involved in GBS virulence, as well as in GBS-PGK surface expression. Mutagenesis of the eveA and eveB genes in GBS resulted in a significant decrease in the surface expression of GBS-PGK, as well as the mutants displayed different GBS phenotypes as compared to the wild type strain, such as decrease in ß-hemolysis, CAMP factor and the production of orange pigmentation. The mutants also showed reduced ability to invade epithelial cells and to resist the phagocytic activity of phagocytic cells within fresh human blood. In order to determine whether the EveA and EveB proteins on GBS surface acting as a binding ligand for GBS-PGK on the bacterial surface, a Far Western Blot was conducted on the purified proteins, EveA and EveB, using the GBS-PGK protein as a probe. Since the EveB protein is a large protein (99.4 kDa) with eight transmembrane domains, the protein was designed to be expressed as two fragments, EveB (up) and EveB (down). The results demonstrate that purified proteins EveA and EveB(up), but not EveB(down) were found to bind the PGK protein on the blot. The immunoreactivity of the purified EveA and EveB proteins with human sera from patients with GBS infection allowed us to determine that EveA and EveB are immunogenic proteins. Using polyclonal rabbit antibodies raised against EveA or EveB proteins in in vitro studies of invasion or opsonophagocytic killing assays, it was investigated that EveA and EveB are not protective. Treating epithelial cells with EveA or EveB(down) prior to infection with GBS inhibited GBS invasion, suggesting EveA and EveB are important in the invasion process. Interestingly, the addition of purified EveA and EveB(down) proteins exogenously to GBS cultures inhibited bacterial growth indicates that EveA and EveB proteins have some antimicrobial activities on GBS. However, their activities in bacterial killing are very low and their minimum inhibitory concentrations (MICs) was measured as 32 and 16 µg/ml, respectively. In this thesis, the effect of polyols in altering GBS phenotypes was studied and the results identified that 1% of erythritol, but not sorbitol or mannitol, was able to stimulate the surface expression of GBS-PGK, increase GBS resistance to phagocytic cells and increase bacterial invasion into HeLa cells suggest that erythritol plays a role in altering GBS phenotypes. These findings show that the presence of GBS in environments enriched with erythritol can affect the nature of the bacteria and transform it from a non-harmful commensal bacterium into a pathogenic one. The data presented in this thesis advances our understanding of the factors in GBS, as well as in the host that are involved in GBS pathogenesis.

  • Subjects / Keywords
  • Graduation date
    Spring 2020
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/r3-9m3h-h806
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.