Pseudomonas aeruginosa is an opportunistic human pathogen which, like many other Gram negative pathogens, employs quorum sensing - regulated virulence factors to establish an infection in its host. Quorum sensing in P. aeruginosa populations is controlled by low molecular weight (hapten) signalling molecules known as homoserine lactones (HSLs). Blocking bacterial communication using antibodies is an attractive strategy for infection control as QS takes a central role in P. aeruginosa infections, and antibodies can recognise their targets with exquisite specificity. There are two well-studied QS circuits in P. aeruginosa- the Las system and the Rhl system, controlled by two autoinducers compounds, 3-oxo-C12-HSL and C4-HSL respectively. Antibodies raised against HSL compounds can reduce the expression of virulence factors controlled by QS circuit and the immunomodulatory effects of 3- oxo-C12-HSL. In order to generate antibodies with high sensitivities against the autoinducer compounds of P. aeruginosa, a panel of HSL compounds was synthesised, conjugated to the carrier protein and used for sheep immunisation. High specificity anti-HSL antibodies were isolated from an immunised sheep antibody repertoire using phage display technology. These phage antibody hits were converted into single chain antibody (scAb) format, which possessed a HuCκ gene for detection and 6x histidine tag for purification. Soluble scAbs expressed in E. coli were purified and characterised using ELISA. Unique clones showing high sensitivity for free HSL compounds were reformatted into sheep-mouse chimeric IgGs, expressed transiently in COS 7 cells and characterised using biochemical assays. These cross-reactive monoclonal antibodies were shown to recognise HSL compounds in low nanomolar concentrations and have the potential to reduce virulence gene expression in P. aeruginosa.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:521346 |
| Date | January 2010 |
| Creators | Palliyil, Soumya |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=136844 |
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