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Title: Structure-function studies of group A streptococcal pilus subunits
Authors: Pointon, Jonathan Anthony
Issue Date: 2011
Publisher: Newcastle University
Abstract: Streptococcus pyogenes (Group A Streptococcus, GAS) is an important Gram positive human pathogen that produces a multitude of virulence factors, including cell-surface pili that were first described shortly before the beginning of this project. Pili in M1 GAS strain SF370 mediate adhesion to human tonsil and skin and are composed of a single major protein subunit (Spy0128) forming the pilus shaft and two minor subunits (Spy0125 and Spy0130), whose roles had not been defined. This thesis reports combined molecular microbiology and protein structure studies that provide insights in to the structures and functions of the M1 GAS strain SF370 pilus subunits. Recombinant Spy0125 (rSpy0125), rSpy0128 and rSpy0130 were purified to apparent homogeneity and low resolution structures were obtained using a combination of biophysical techniques. Novel intra-molecular isopeptide bonds discovered in the Spy0128 crystal structure, reported by others during this project, were examined here for their affects on GAS adhesion. This was achieved by combining site-directed and allele replacement mutagenesis to introduce point mutations into the spy0128 gene in the GAS chromosome. Comparison of parent and mutant strains showed that these bonds were not essential for adhesion of GAS to human keratinocytes in vitro, though the pattern of adhesion appeared altered. Most of the work in this project focused on Spy0125, which was first localized at the tip of the pilus and shown to act as the adhesin. Allele replacement mutagenesis confirmed that the adhesin resided within a stable c. 50 kDa polypeptide corresponding to the Cterminal 2/3 of intact Spy0125. A recombinant version of this region, rSpy0125-CTR, was produced and its high resolution crystal structure determined. In addition to internal intra-molecular bonds similar to those recently found by others in Spy0128, the rSpy0125-CTR structure revealed an internal thioester bond between a Cys and a Gln residue that is unprecedented outside of complement and complement-like proteins. Whereas current paradigms of bacterial-host interactions suggest non-covalent forces are involved, the presence of a thioester in Spy0125 reveals for the first time that strong covalent forces may also play a role.
Description: PhD Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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