Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.727472
Title: Characterization of the interactions of the Streptococcus pneumoniae toxin, pneumolysin, with soluble molecules of the immune system
Author: Faraj, Bayan Hama Amin
ISNI:       0000 0004 6424 9198
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2017
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Abstract:
Pneumolysin (Ply) is a key virulence factor of the bacterium Streptococcus pneumoniae (Pneumococcus). Major functions include forming pores in mammalian cell membranes and activating the complement cascade to divert the host’s immune system. The aim of this thesis was to investigate these processes at the molecular level to understand how Ply facilitates disease by the pneumococcus. Previous studies have suggested that Ply interacts with various soluble molecules of the immune system, including L-ficolin and IgG. These interactions activate complement via the lectin and classical pathways, respectively. In this thesis I have demonstrated that Ply does not interact with either native serum L-ficolin or recombinant human L-ficolin produced in Chinese hamster ovary cells. The previous erroneous report probably arose as a result of contamination of Ply preparations with L-ficolin ligands. Investigation of binding between Ply and IgG showed that Ply binds to IgG2, IgG3 and IgG4 but not to IgG1. Binding is mediated through interactions between domains 1-3 of Ply and the Fab region of the IgGs. An additional aim of this thesis was to investigate pore formation by Ply. The crystal structure of Ply, determined in our group, showed that Ply monomers in the crystal pack together similar to the way in which they are likely to assemble on the cell surface prior to pore formation. Based on the structure, a series of mutations were created to disrupt packing between Ply monomers during pre-pore and pore formation. The activities of two of the mutants, Asp205Arg and Asn339Arg were completely abolished and most of the mutants had greatly reduced activities compared to wild-type Ply indicating that these residues play important roles during pore formation. Interestingly, electron microscopy showed that Ply Asp205Arg forms chain like structures on membranes but cannot form circular pores or arcs. Thus although monomers still self-associated they could not kill cells. By contrast, Ply Asn339Arg, binds to the membrane but does not oligomerize. In further work, crystal structures of the membrane-binding domain of Ply revealed conformational changes in a Trp rich-loop at the base of the toxin involved in membrane binding. These changes promote new packing interactions between Ply monomer thereby promoting oligomerization on the membrane. Finally, I investigated the structural changes of the membrane by spectroscopic monitoring of optically trapped vesicles. The inelastic back-scattered light was monitored from a single liposome, held by optical tweezers and exposed to Ply. Ply binding increased the membrane fluidity due to a decrease in the short-range order of the lipid molecules in the bilayer. Analysis of a series of point mutants suggests that these changes are caused by association of Ply monomers during formation of the pre-pore, prior to insertion across the membrane.
Supervisor: Wallis, Russell ; Andrew, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.727472  DOI: Not available
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