The characterisation of the cell surface of Staphylococcus aureus in the search for new therapeutic targets

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Staphylococcus aureus is a Gram-positive pathogen which causes a wide range of afflictions including endocarditis, osteomyelitis, cellulitis, toxic shock syndrome, and necrotising pneumonia. S. aureus is a leading cause of hospital-acquired infections and has rapidly acquired resistance to multiple antimicrobials. As such, it was deemed a serious threat by the Centers for Disease Control and Prevention (CDC) in 2013 indicating urgent attention is required to control this pathogen. Currently there are no efficacious vaccines available to treat infections caused by S. aureus. With resistance being noted against every class of antibiotic currently available, the development of an alternative therapeutic would not only relieve morbidities, mortalities, and the associated economic burden, but also reduce the selective pressures that drive antibiotic resistance. To better understand how S. aureus interacts with the human host and presents antigens that interact with key host cell receptors, a better understanding of which proteins are displayed on the cell surface is required. This dissertation presents an analysis of the surface proteome of S. aureus and describes several potential novel adhesins. Enzymatic cell shaving and surface protein biotinylation were used to catalogue proteins on the cell surface and identify regions within molecules that are surface accessible. Our approaches included methods that maintained protein size context (SDS-PAGE), providing an insight into the extent of surface protein processing. We also characterised heparin-binding proteins in S. aureus and interrogated the data in light of our surface proteome studies. This approach enabled us to gain insight into novel binding characteristics used by surface-accessible proteins that could not be predicted using reverse vaccinology and other hypothesis-directed approaches commonly used to develop potential vaccine candidates. Ascertaining the repertoire of heparin-binding proteins was considered important as these proteins are bacterial virulence factors that facilitate adherence, colonisation, and invasion of target host cells. By coupling these data with other proteomic and bioinformatics techniques, a number of proteins of interest were identified. This includes Elongation Factor Tu, which was found to be surface exposed and highly processed, a finding that has not been seen before in S. aureus. The data presented in the following chapters contributes significantly to the rapidly evolving field of S. aureus proteomics. These data will aid in the development of future therapeutic strategies and highlights a number of proteins for further therapeutic investigation.
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