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Title: Relationship of structure to function in the pore-forming toxin pneumolysin from Streptococcus pneumoniae
Author: El-Rachkidy, Rana Georges
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2003
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Pneumolysin is an important virulence factor produced by the human pathogen Streptococcus pneumoniae. It belongs to the family of cholesterol dependent cytolysins (CDCs) that damage the target cell membrane, by forming large oligomeric pores of 30 to 80 toxin monomers, where each monomer is thought to contribute at least two p-hairpins. A panel of mutations was done in the two putative transmembrane region of pneumolysin TMH1 and TMH2 located in domain 3 of the toxin monomer, and believed to be lining the pore lumen. The generated mutants exhibited different levels of haemolytic activity, particularly the single mutation W278F, W278D, and the triple mutation (D257N-E258Q-E260Q), largely impaired the haemolytic activity of the wild-type toxin. These mutant toxins along with a previously made lytic deficient mutant W433F were subjected to further studies. Circular dichroism analysis done with those mutants showed that the secondary structure of the native toxin was conserved. The kinetics of release of calcein from liposomes along with the kinetics of lysis of erythrocytes exposed to these mutants was substantially slower than that of the wild-type toxin. Pneumolysin and other CDCs induced pores were studied on model systems like lipid bilayer and liposomes. In this thesis, I demonstrated the formation of pores by pneumolysin on the membrane of a 'real' cell by using the patch-clamp technique. Pneumolysin induced heterogeneous pore on either side of the membrane, of different conductance states, classified as small, medium and large. A stepwise increase in current was observed with early appearance of small conductance channels followed by larger ones. The mutant toxins generated in this work and W433F were also tested with patch clamping. They formed pores of various conductance states with a decrease in the occurrence of large channels, in comparison to the wild-type.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available