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|Title:||Structure and function of pneumolysin, the thiol-activated toxin of Streptococcus pneumoniae.|
|Authors:||Owen, R. H. G. (Richard H. G.)|
|Presented at:||University of Leicester|
|Abstract:||Streptococcus pneumoniae, or the pneumococcus, is an important pathogen of man. Pneumolysin is a cytolytic toxin produced by the pneumococcus, that binds to all eukaryotic cells tested. The aim of this research project was to locate the cell- binding region of pneumolysin. Two separate strategies were used to locate the cell- binding region of pneumolysin. One approach was to produce a gene library which contained single, random, point mutations throughout the pneumolysin gene. The products of the experiment were screened for mutants which showed reduced haemolytic activity as it was thought that mutants with reduced cell-binding activity would also possess reduced haemolytic activity. The mutants which were found to possess reduced haemolytic activity were to be tested for cell-binding activity. The experiment was performed but no phage were produced. However, by using the same approach, a colleague subsequently produced mutated phage which possessed reduced haemolytic activity. None of these have yet been assayed for cell-binding activity. The other approach was to produce truncated versions of the pneumolysin protein and assay their cell-binding activity. Using this approach it was found that deletion of 20 residues from the C-terminus of pneumolysin completely abolished cell- binding activity. Molecules with smaller C-terminal deletions had much lower cell-binding activity than the intact molecule. Deletion of six residues from the C-terminus of pneumolysin reduced cell-binding activity by 98%. Using site-directed mutagenesis, a proline residue in the C-terminal region of pneumolysin was replaced with serine. The resultant full-length molecule showed a reduction in cell- binding activity of 90% when compared to the wildtype molecule. The results showed that residues in the C-terminal region of pneumolysin are necessary for cell-binding activity.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biology|
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