Date of Award

2011

Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy

Department

Biological Sciences

First Advisor

Dr. Aidan Coffey

Abstract

The work presented in this thesis focuses on the purification, characterisation and practical applications of an anti-staphylococcal lysin (LysK) and its single-domain truncated derivative (CHAPk) against pathogenic staphylococci including meticillin resistant Staphylococcus aureus (MRSA). Both proteins were purified to greater than 90% homogeneity. Affinity chromatography was applied as the optimum method for LysK purification and ion-exchange chromatography was successfully applied to CHAPk. The lytic spectrum of LysK was investigated by zymogram analysis where the enzyme exhibited strong activity against a range of staphylococci regardless of their origin, sequence type (ST) or antibiotic resistant profile. This analysis included all S. aureus sequence types in existence in Ireland from the National MRSA Reference laboratory, Dublin and exopolysaccharide (EPS) producing S. aureus strains associated with bovine mastitis. Subsequent studies revealed that the LysK enzyme was unstable and prone to aggregation which ultimately restricted its development for possible applications in in vitro and in vivo experiments. CHAPk , the truncated single-domain derivative of LysK was found to be a stable, highly active, highly soluble enzyme. Purification using cation exchange chromatography regularly yielded up to 10 mg of pure, soluble, active protein per litre of E. coli culture. The lytic spectrum of CHAPk, not only included all staphylococci that were susceptible to the native three-domain LysK protein but also extended to members of the genera Micrcococcus, Streptococcus, Nesterenkonia, Arthrobacter, Leuconostoc and Camobacterium. Characterisation of CHAPk over a range of parameters and conditions revealed the enzyme was active from pH 6 to 11 with an optimum activity at pH 9; from 5°C to 40° C, with an optimum activity at 15°C and also maintained activity in the presence of 0 to 300 mM NaCl. In addition CHAPk was analysed in the presence of a variety of inhibitors and the effect of repeated freeze-thaw cycles and storage at 4°C and -80°C was also recorded. When culture lysis by CHAPk and the metalloendopeptidase lysostaphin were compared over a concentration range of 2.5 to 10 µg/ml using live S. aureus cells for 5 min at 37°C, CHAPk gave rise to greater turbidity reduction indicating that it works more rapidly than lysostaphin. Further studies were performed to investigate the practical applications of CHAPk for the control and prevention of staphylococcal infection. In one such study CHAPk was shown to rapidly disrupt and eliminate mature biofilms of S. aureus within an hour. CHAPk was not only able to disrupt mature biofilms but was also able to completely prevent biofilm formation. CHAPk was explored as a spray decolonisation agent to remove S. aureus from skin and the enzyme removed 99% of S. aureus in 30 min. An in vivo trial was performed using CHAPk with an In Vivo Imaging System (IVIS) and BALB/c mice. Treatment with CHAPk successfully eliminated a lux-labeled S. aureus strain from the nares of the inoculated animals. Finally, in silico analysis was undertaken on the enzyme resulting in the predicted three-dimensional structure of the CHAPk protein. The overall results in this thesis provide new insights into the novel anti-staphylococcal enzyme CHAPk tind indicate it has significant potential for development as an effective antibacterial agent against pathogenic staphylococci including MRSA.

Access Level

info:eu-repo/semantics/openAccess

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