Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.518652
Title: Identification of MRSA-specific peptides and expression of peptide containing recombinant proteins for targeted photodynamic therapy
Author: Vince, Rebecca
Awarding Body: The University of Hull
Current Institution: University of Hull
Date of Award: 2009
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Abstract:
Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of causing infection at multiple sites and is one of the predominant organisms found in chronic wounds, such as venous ulcers. Furthermore, there is an ever-increasing prevalence of multi-drug resistant strains, such as methicillin-resistant S. aureus (MRSA) that can contribute to the non-healing and infection of wounds as well as other life-threatening complications. The increase in bacterial resistance necessitates the development of alternative modes of bacterial targeting and elimination whilst avoiding collateral damage to host cells and commensal bacteria. One such modality is photodynamic therapy (PDT), whereby the activation of a photosensitiser by light in the presence of oxygen produces reactive oxygen species to elicit bacterial cell death. PDT has previously been shown to be effective against a wide range of Gram-positive and Gramnegative bacteria and furthermore selective targeting of the photosensitiser directly to the pathogenic bacteria is likely to improve the efficacy of this treatment.The FliTrx (TM) bacterially-displayed random peptide library (Invitrogen), displaying 12-mer peptides in a conformationally constrained manner on the surface of E. coli was screened by panning against UV-inactivated MRSA. Panning was also performed against S. aureus and S. epidermidis in a similar manner. Clones (n = 20) were then randomly selected, DNA sequenced and peptide sequences analysed. No obvious consensus sequence or repeat sequences were observed. Two peptides selected from panning against MRSA were commercially synthesised (peptide19 and peptide12) as both cyclic and linear constructs with N-terminal biotin for characterisation of binding by flow cytometry. Furthermore, one peptide (peptide12) was synthesised as both a cyclic and linear construct replacing the biotin with C-terminal FITC for direct characterisation of peptide binding by flow cytometry. Binding of the cyclic construct was observed against 3 different strains of MRSA, with the greatest binding observed against the strain of MRSA originally panned against. No binding of the linear construct was observed. Low/no reactivity was observed with other Staphylococcal sp, Gramnegative bacteria and a mammalian cell line demonstrating apparent specificity for MRSA.Peptide sequences of interest were also cloned in-frame, situated within the full length thioredoxin sequence, so as to reflect the original display format, into an E. coli based expression vector. Recombinant protein expression was optimised and the protein extracted, purified and characterised by Western blotting. The porphyrin photosensitiser (5-(4-isothiocyanatophenyl)-10,15,20-tris-(4-N-methylpyridiniumyl) porphyrin trichloride) was conjugated to recombinant protein P12 at a molar ratio of 5:1 and the in vitro cytotoxic effect of the resulting bioconjugate was determined against MRSA, E. coli and keratinocytes. Pilot data demonstrated a 66% reduction in MRSA growth following incubation and irradiation with the bioconjugate in comparison to nonirradiated control cells. The conjugated porphyrin gave higher levels of cytotoxicity than equivalent concentrations of unconjugated capped porphyrin. No toxicity of the conjugate or the capped porphyrin against MRSA was observed when light of the correct wavelength was not given. Additionally no/little cytotoxic effect against E. coli or mammalian keratinocytes was observed.Additionally, binding of two other purified recombinant proteins (P5 and P19) to MRSA, E. coli and keratinocytes was assessed by flow cytometry. Both recombinant proteins were observed to bind MRSA in comparison to E. coli and keratinocytes.Furthermore, one sequence (P5) was expressed as an additional GFP fusion protein construct to allow direct characterisation of recombinant protein binding. A very small increase in binding was observed with an increasing concentration of recombinant protein against MRSA, E. coli and keratinocytes.In summary, this thesis has described a system to isolate and characterise novel peptides with binding specificity against MRSA. Furthermore, it has demonstrated the feasibility of expressing recombinant proteins and conjugation to a porphyrin photosensitiser for targeted PDT against pathogenic bacteria whilst limiting damage to the host, providing an alternative to antibody-based targeting in microbiology.
Supervisor: Greenman, John (supervisor) Sponsor: Smith & Nephew (sponsor)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.518652  DOI: Not available
Keywords: Biological sciences
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