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Title: Analysis of Staphylococcus aureus virulence determinants
Author: Connolly, John
ISNI:       0000 0004 5921 6607
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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The success of the pathogen Staphylococcus aureus lies in its array of virulence determinants, which enable pathogenesis. The recent identification of novel S. aureus virulence determinants led to the hypothesis that there are more to be found. In silico analysis of the S. aureus genome identified three staphylococcal superantigen-like proteins (SSL12, SSL13 & SSL14), incorrectly annotated in the genome database as hypothetical proteins. Production of recombinant SSL12, 13 & 14 proteins gave a low yield of soluble protein, which precluded biochemical analysis. A genetic approach was taken and a triple gene deletion mutant constructed. No role for the 3 SSLs was found in an in vivo infection model, or in in vitro phagocytosis assays. However, a subtle reduction in growth in human blood, associated with the cellular component of blood was seen when compared with the wild-type. A genome-wide library of 1,920 strains each with a separate gene disrupted by a transposon (Tn) insertion was screened on human blood agar. Both the purine (purA and purB) and tetrahydrofolate (THF; pabA) synthesis pathways were found to be important for growth on human blood, but, in the case of the pabA disrupted strain, not on human plasma. THF is a single carbon donor/acceptor in many S. aureus biosynthesis pathways, and its synthesis is the target of sulphonamide antibiotics. The human blood phenotype for pabA was linked to dTTP synthesis, which is formed via a THF-dependent pathway, or a THF-independent pathway requiring the enzyme Tdk. As the pabA mutant can grow on human plasma it was hypothesised that Tdk is inhibited by a factor in the cellular component of blood, which leads to a requirement for dTTP. This suggests that the activity of sulphonamide drugs is the result of inhibition of THF coupled with the inhibition of Tdk by an as yet unknown factor present in human blood.
Supervisor: Foster, Simon J. ; Whyte, Moira K. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available