Date of Award
Doctor of Philosophy
Dr. Aidan Coffey
The work presented in this thesis focuses on harnessing the bactericidal potential of bacteriophage (phage) encoded peptidoglycan hydrolase enzymes to combat the significant pathogen Staphylococcus aureus. In order to identify novel peptidoglycan hydrolases lytic against S. aureus, the complete genomes of two staphylococcal temperate Siphoviridae bacteriophages, DW2 and CSl, were sequenced and annotated. Screening of these genomes identified genes for several putative peptidoglycan hydrolases including two endolysins and three virion-associated peptidoglycan hydrolases. The identification of putative genes relating to virulence and host fitness lends to the understanding of the role played by such temperate phages in the evolution of this important pathogen. In silico analysis of the genome sequence also identified a putative serine recombinase and a novel Bcg1-like restriction modification system which provide potential templates for development of novel molecular tools. With the aim of producing novel recombinant anti-staphylococcal proteins, the endolysin and tail-tip peptidoglycan hydrolase genes of phage DW2 were cloned into an Escherichia coli expression system and their gene products were demonstrated to have staphylolytic activity. In addition, some of the recombinant proteins produced demonstrated lytic activity against a methicillin resistant S. aureus strain and an exopolysaccharide producing S. aureus strain. The tail-associated peptidoglycan hydrolase gene from phage DW2, named HydDW2, was experimentally verified to contain an intron. This is the first report of an intron in an S. aureus temperate phage and also the first report of an intron in a phage tail-associated peptidoglycan hydrolase gene. The gene product was demonstrated to have a broad lytic spectrum with activity against selected Gram positive and Gram negative bacteria. Biochemical and structural analysis was also performed on the recombinant CHAPk enzyme derived from the LysK endolysin of staphylococcal phage K. Work presented in this thesis demonstrated that CHAPk was able to disrupt a mature staphylococcal biofilm in under four hours. In addition, it was shown to prevent staphylococcal biofilm formation. These results are significant because bacteria in biofilms are more recalcitrant to antibiotic treatment and staphylococcal biofilms are often the cause of recurring infections. In addition, the crystal structure of CHAPk was elucidated and lead to greater insight into the catalytic mechanism of this enzyme. Most notably a calcium ion was found to be coordinated in close proximity to the active site and site directed mutagenesis indicated the essential involvement of this ion in catalytic activity of the enzyme. This information gives insight into the fundamental reaction mechanism of the enzyme and provides invaluable information for protein engineering of CHAP (Cysteine, histidine-dependent amidohydrolase/peptidase) domain containing enzymes.
Keary, Ruth, "Characterisation of the Enzybiotic Potential of Selected Staphylococcus Aureus Bacteriophages" (2014). Theses [online].
Available at: https://sword.cit.ie/allthe/472