| Summary: | Burns represent one of the most devastating forms of injury with infection complications representing the highest risk of mortality. The primary objective of the Bacteriosafe project was the development of a smart wound dressing that would respond to the presence of bacteria in burn wounds. The basis of this sensing system employed the use of phospholipid vesicles, containing a self-quenchable fluorescent dye. These vesicles mimic the eukaryotic cell membrane and as such are susceptible to bacterial cytolytic factors which lyse the vesicles, generating an observable and measurable fluorescent response. My primary role in this project was to identify the vesicle lysing agents secreted from the two most frequent burn wound colonisers, Staphylococcus aureus and Pseudomonas aeruginosa. We identified the small amphipathic alpha helical peptide toxins from S. aureus and glycolipid molecules derived from P. aeruginosa as the agents responsible for vesicle lysis. The identification of these molecules led to the development of two novel phenotypic assays designed to measure these important virulence factors, as discussed in chapter 3 and 4. In chapter 5 we examined the role of toxic shock syndrome toxin-1 (TSST-1) in repressing global exoprotein expression. Our results demonstrate that TSST-1 does not repress toxin secretion and strains expressing TSST-1 retain their ability to lyse vesicles. In chapter 6 we explored the use of subinhibitory oxacillin in inducing the alternative penicillin binding protein 2a (PBP2a) in community-acquired methicillin resistant S. aureus (CA-MRSA) strains to down-regulate toxicity. Previous work in the Massey lab demonstrated that the expression of the mecA gene, which encodes PBP2a, resulted in reduced toxicity in hospital-acquired (HA) -MRSA. CA-MRSA strains are considered highly toxic and have a considerably lower level of PBP2a expression. Treatment of CA-MRSA strains with subinhibitory oxacillin did result in a down-regulation of some toxins but also the up-regulation of others, highlighting the pleiotropic effect oxacillin had on virulence regulation. In chapter 7 we developed an approach that uses the genome sequences of a set of related clinical S. aureus strains to identify novel virulence loci by associating genetic polymorphisms with specific virulence phenotypes using a genome wide association study (GWAS). This analysis resulted in the identification of four novel loci which when mutated lead to a reduction in toxicity. We demonstrate that the GWAS approach is an effective method in identifying candidate SNPs which may be important in altering virulence but do highlight limitations of this approach, primarily the generation of false positives.
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