Staphylococcus pseudintermedius is the major cause of the common canine skin disease, pyoderma, and is a zoonotic pathogen of humans. Multidrug resistant strains of S. pseudintermedius have emerged and are spreading globally leading to decreased therapeutic success. The development of novel therapeutics is hindered by the lack of understanding of critical host-pathogen interactions mediating S. pseudintermedius colonization and pathogenesis. For the major human pathogen Staphylococcus aureus, interactions with host fibrinogen play a fundamental role in pathogenesis. The aim of the current study was to genetically and functionally characterise 2 cell wall-associated proteins of S. pseudintermedius, SpsD and SpsL, which mediate binding to multiple host extracellular matrix proteins including fibrinogen and fibronectin. DNA sequencing of the A- (ligand binding) domains of spsD and spsL genes for 37 phylogenetically diverse isolates revealed a highly conserved sequence for SpsL (97.1% derived amino acid identity), in contrast to more extensive variation for SpsD (76.7% derived amino acid identity). Further, recombination events were identified to have contributed to the evolution of spsD diversity. Functional analysis with gene deletion mutants of S. pseudintermedius strain ED99, constructed in the current study, demonstrated that SpsL is a major cell wall-associated fibrinogen-binding protein with enhanced affinity for canine fibrinogen. Using recombinant chains of fibrinogen it was determined that SpsL binds to the α-chain of fibrinogen similar to clumping factor B (ClfB) of S. aureus. However, ELISA and surface plasmon resonance analyses of recombinant truncated derivatives of SpsL indicated that the predicted ligand-binding N2N3 subdomains of the A-domain of SpsL are not sufficient for high-affinity fibrinogen-binding suggesting that either additional domains or post-translational modifications are required for fibrinogen-binding. Furthermore, development of a murine skin infection model allowed an investigation of the contribution of SpsD and SpsL to pathogenesis revealing a role for SpsL in focal abscess pathology. Overall these studies have provided new insights into the diversity, function and therapeutic potential of S. pseudintermedius fibrinogen-binding proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:735701 |
| Date | January 2016 |
| Creators | Richards, Amy Claire |
| Contributors | Fitzgerald, Jonathan ; Lengeling, Andreas |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/25715 |
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