Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, it gains access to the bloodstream and causes septicaemia and subsequently meningitis. T-cell stimulating protein A (TspA) is an immunogenic, conserved, T -cell and B-cell stimulating protein of N meningitidis that is required for optimal adhesion to human cells. The role of TspA in the adhesion process is thought to be indirect since it is predicted that the N-terminus is localised to the periplasmic space with an inner membrane- spanning domain linking it to a cytoplasmic C-terminal domain; the aim of this study was to identify proteins within the meningococcal envelope that interact with TspA. Four overlapping recombinant fragments of TspA were expressed and purified as fusion proteins with the pGEX-2T-encoded glutathione-S- transferase protein and pQE70. A receptor activity-directed affinity tagging protocol was employed to identify TspA-interacting meningococcal proteins. Interactions between TspA and candidate proteins were investigated further using ELISA and surface plasmon resonance. Four putative TspA-interacting proteins were identified: PilQ and PilT (components of the type IV pilus machinery); the major outer membrane protein, PorA, and the protein chaperone, ClpB. Furthermore, the portion of TspA responsible for interaction with PorA was confirmed to be the N- terminus. Together, findings show that periplasmic domains of TspA interact with several outer membrane proteins which have a key role in meningococcal pathogenesis and adhesion to host cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:556164 |
| Date | January 2011 |
| Creators | Ahmed, Nader Wahba Abdelrahman |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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