Horsham, Matthew Robert
Structural and functional analysis of pneumococcal histidine triad D from Streptococcus pneumoniae.
PhD thesis, University of Glasgow.
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The pathogenic bacteria Streptococcus pneumoniae is one of the major causes of morbidity and mortality in humans in the world today. A Gram-positive facultative anaerobe, under natural conditions it exists as a commensal bacteria residing in the nasopharynx. Upon invasion of the body, S. pneumoniae can cause a number of diseases, which range in severity from acute otitis-media to pneumonia, septicaemia and meningitis.
The main virulence factor of S. pneumoniae has been identified as the polysaccharide capsule, which coats the outside of the cell. S. pneumoniae can be categorised into 92 distinct serotypes based on capsular composition. Current available vaccines utilise a mixture of polysaccharides from the most prevalent capsular serotypes, or capsular polysaccharide conjugated to a carrier protein. Vaccine coverage is therefore serotype specific. Furthermore, vaccine efficiency is lower in those groups most at risk of infection; namely infants, the elderly and the immuno-compromised. As a result investigation is ongoing into a next generation of protein-based vaccine that can provide increased coverage and efficiency.
A novel family of proteins termed Pneumococcal Histidine Triad (Pht) proteins were identified from a whole genome antigen-screen as potential candidates for inclusion in a novel protein-based pneumococcal vaccine. Animal models of infection have shown that immunisation with the Pht protein Pneumococcal Histidine Triad D (PhtD) confers protection against invasion of S. pneumoniae.
PhtD was cloned, expressed, purified and subjected to crystallisation trials in an attempt to uncover the function of PhtD through determining the protein structure by macromolecular X-ray crystallography, which yielded some rudimentary crystallographic data. Biophysical analysis of PhtD using a variety of techniques including limited proteolysis and circular dichroism revealed that PhtD exclusively bound the divalent-cation Zn2+ and that Zn2+-binding induced a major conformational change in the protein structure, which proved to be a reversible process. A rationalised, targeted analysis of PhtD protein structure by limited proteolysis, Nuclear Magnetic Resonance (NMR), N-terminal sequencing and mass-spectrometry revealed localised, ordered regions of structure within the protein sequence that were highly stable. These identified protein fragments were subsequently cloned, expressed, purified and subjected to crystallisation trials. Due to their smaller and more ordered nature, it was postulated that these PhtD fragments may prove more readily crystallisable than the full-length molecule. Initial crystals have been obtained for these protein fragments and are being optimised to improve crystal size and quality. Evaluation of the PhtD proteolysis products by Western blotting with anti-PhtD antibody has revealed the dominant epitope for the PhtD protein, localised to a 15 kDa region in the C-terminal half of the PhtD protein sequence. This could be a major advancement in development of a protein based vaccine as only the 15 kDa epitope-containing region need be included in order to elicit an antibody reaction. Furthermore, the small size of the protein fragment is highly conducive to structural determination by a variety of methods. This 15 kDa epitope fragment has been cloned into an expression plasmid allowing recombinant protein expression in order to investigate further.
Additionally, as training for handling of PhtD X-ray diffraction data, macromolecular X-ray crystallography was attempted with a variety of different proteins from Gram-positive bacteria. Two proteins -a novel cis-trans isomerase PpmA from S. pneumoniae, and the transketolase TktA from Lactobacillus salivarius- were successfully crystallised. Diffraction quality crystals of TktA were grown that produced X-ray diffraction to 2.3Å resolution. The structure of TktA was successfully determined using the molecular replacement method. Diffraction quality crystals of PpmA were grown that show X-ray diffraction to 2.5Å resolution, and optimisation of crystallisation conditions should yield better X-ray diffraction, allowing the structure of PpmA to be determined. The data pertaining to these proteins is also included as part of this thesis.
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