Staphylococcal cell wall associated proteins : Characteristics and host interactions

Detta är en avhandling från Stockholm : Karolinska Institutet, Department of Laboratory Medicine

Sammanfattning: Staphylococcus aureus and Staphylococcus epidermidis are members of the same family and share several features. S. aureus causes many diseases ranging from mild skin infections to deep tissue infections and septic shock. S. epidermidis infections are generally milder, but affect foreign bodies such as indwelling catheters. In this study both novel and already characterised plasma and extra cellular matrix binding proteins are characterised and studied as targets for vaccination and passive immunisation. A peptide from the S. aureus fibronectin-binding protein, FnBP, was displayed on the surface of a plant virus, CPMV, which is non-infectious in mammals. This construct displays the fibronectinbinding region of the protein, but lacks binding capacity. This prevents undesired host interactions and antibodies directed against ligand-induced binding sites. Antibodies directed against FnBP-CPMV opsonised S. aureus and enhanced phagocytosis in vitro. The FnBP-CPMV was used to immunise rats subsequently subjected to a S. aureus septic arthritis/endocarditis model. Vaccination protected against dissemination from the infected joints to heart valves, and passive administration of generated antibodies protected mice from bacterial infection. FnBP has previously been demonstrated to have importance for internalisation into human cells, such as endothelial cells. I have also demonstrated that the internalisation rate of S. aureus was much lower in endothelial cells from tissue compared to the corresponding cultured cells. The S. epidermidis fibrinogen-binding protein Fbe was used to generate specific antibodies that opsonised bacteria. The antibodies were protective against mouse sepsis, and enhanced macrophage phagocytosis of opsonised S. epidermidis. Human serum pools enriched for antibodies directed against Fbe also enhanced opsonophagocytosis, which demonstrates that Fbe naturally induces a functional immune response. Searching the S. epidermidis RP62A genome for open reading frames with sortase recognition domains and signal sequences resulted in the identification of six previously uncharacterised cell wall-associated proteins. The novel proteins were designated SesB, C, E, G, H and 1, as for "Staphylococcus epidermidis surface protein". Other proteins recognised were Fbe, Bap-1 homologue, accumulation-associated protein and a cell division protein. The proteins were cloned and antibodies were generated. Of all antibodies, only those directed against Fbe and SesI showed opsonising activity in a macrophage ingestion assay. Collectively, I have demonstrated the vaccine potential of S. aureus FnBP, and S. epidermidis Fbe. The role of FnBP for internalisation is not pronounced in fresh human endothelial cells in biopsies, and the internalisation rate of S. aureus is lower compared to what it is in cultured cells. Furthermore, six previously uncharacterised cell wall associated proteins from S. epidermidis were preliminary characterised. The novel protein SesI displayed opsonic activity and is of interest for further investigation as a vaccine candidate.

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