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Phage Therapy and Development of Delivery Systems for Gram-Positive Phage Endolysins" (2018)

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Phage Therapy and Development of Delivery Systems for Gram-Positive Phage Endolysins Munster Technological University SWORD - South West Open Research Deposit PhDs Science 9-2018 Phage Therapy and Development of Delivery Systems for Gram- Positive Phage Endolysins Jude Ajuebor Department of Biological Sciences, Cork Institute of Technology Follow this and additional works at: https://sword.cit.ie/scidiss Part of the Biology Commons Recommended Citation Ajuebor, Jude, "Phage Therapy and Development of Delivery Systems for Gram-Positive Phage Endolysins" (2018). PhDs [online]. Available at: https://sword.cit.ie/scidiss/4 This Doctoral Thesis is brought to you for free and open access by the Science at SWORD - South West Open Research Deposit. It has been accepted for inclusion in PhDs by an authorized administrator of SWORD - South West Open Research Deposit. For more information, please contact [email protected]. Phage therapy and development of delivery systems for Gram-positive phage endolysins A thesis submitted to Cork Institute of Technology for the degree of Doctor of Philosophy By Jude Ajuebor, B.Sc. Supervisor: Prof. Aidan Coffey Department of Biological Sciences, Cork Institute of Technology September 2018 Acknowledgements Foremost, I would like to thank my supervisor Prof. Aidan Coffey for his support, guidance and devotion throughout the duration of my PhD. I would also like to thank the Lecturing and Technical staff in the Department of Biological Sciences for their support and advice. My sincere appreciation to those scientists that graciously contributed invaluable resource and tools aiding me throughout this work, Dr. Daniel Burke, Dr. Andrey Shkoporov, Dr. Angela Back, Dr. Mark J. Van Raaij, Dr. Antonio Pichel Beleiro, Dr. Horst Neve, Dr. Olivia McAuliffe, Prof. Jim O'Mahony and Prof. Colin Hill. A special gratitude to all the postgrad past and present for their friendship and support during the duration of my PhD, in particular Marcel de Kruij, Rodney Govender, Colin Buttimer and Caiomhe Lynch. A special thanks also to my friends Adonai Djankah and Carlota Marquez Grana for their advice and support. Finally, to my family, special thanks for your understanding and continued support during the duration of my academic pursuit. i Thesis Abstract This thesis focussed on Gram positive phages and their endolysins. Here, two similar kay-like staphylococcal phages B1 (vB_SauM_B1) and JA1 (vB_SauM_JA1) were isolated from a commercial therapeutic phage mix. Their host range was established on the Irish National MRSA bank, which included twenty one sequence types in addition relevant control strains. Based on this, distinct phages were identified and subjected to genome sequencing. The sequences were compared with the sequence of phage K (vB_SauM_K), which was also determined in this work. All three phages had a genome size of at least 139 kb, although some key differences were identified between each. The new phages B1 and JA1 possessed double stranded DNA and generally had a broader host range than phage K. A comparative genomic analysis on the phage genomes identified several (open reading frames) ORFs that were absent in the genome of phage K but present in genomes of phages B1 and JA1. One of the cloned genes from phage K was shown to encode a protein for the receptor-binding- protein and this protein was demonstrated to slightly inhibit phage adsorption. The other cloned gene encoded the phage endolysin and this peptidoglycan hydrolase were identical across all three phages and thus, the CHAPk endolysin of phage K was chosen to demonstrate the application of the endolysin for the control of staphylococci in milk. A two-log reduction in staphylococcal numbers in milk was observed. When the endolysin was introduced into a lactococcal secretion system using the pNZ8048 vector, detectable secretion was successfully demonstrated. Simultaneously, a Clostridium difficile phage endolysin, an amidase, was also cloned into the same secretion system with successful secretion also being demonstrated. In addition, this latter endolysin was also secreted from a recombinant E. coli strain, suggesting potential applications for delivery of the endolysin to the intestine from a hypothetical probiotic E. coli strain. ii List of publications Ajuebor, J.; McAuliffe, O.; O’Mahony, J.; Ross, R. P.; Hill, C.; Coffey, A. Bacteriophage endolysins and their applications. Sci. Prog. 2016, 99, 183–199, doi:10.3184/003685016X14627913637705. Hathaway, H.; Ajuebor, J.; Stephens, L.; Coffey, A.; Potter, U.; Sutton, J. M.; Jenkins, A. T. A. Thermally triggered release of the bacteriophage endolysin CHAPKand the bacteriocin lysostaphin for the control of methicillin resistant Staphylococcus aureus (MRSA). J. Control. Release 2017, 245, 108–115, doi:10.1016/j.jconrel.2016.11.030. Ajuebor, J.; Buttimer, C.; Arroyo-moreno, S.; Chanishvili, N.; Gabriel, E. M.; Mahony, J. O.; Mcauliffe, O.; Neve, H.; Franz, C.; Coffey, A. Comparison of Staphylococcus phage K with close phage relatives commonly employed in phage therapeutics. Antibiotics 2018, 7, 37, doi:10.3390/antibiotics7020037. List of conferences attented Ajuebor, J., McAuliffe, O., O’Mahony, J., Ross, R.P., Hill, C., Coffey, A. (2015). Expression and secretion of staphylococcal phage endolysin CHAP in a Lactococcus lactis cheese starter. 44th Annual Food Research Conference. Cork, Ireland. Dec 14 2015. Ajuebor, J., McAuliffe, O., O’Mahony, J., Ross, R.P., Hill, C., Coffey, A. (2016). Engineering a phage endolysin against Clostridium difficile for delivery into the gastrointestinal tract. EMBO conference on the Viruses of Microbes. Liverpool, U.K. July 18-22 2016. Ajuebor, J., Keating, A., Djankah, A., McAuliffe, O., O’Mahony, J., Ross, R.P., Hill, C., Coffey, A. (2017). Host range and comparative genomics analysis on three similar Myoviruses. Phages 2017: Bacteriophage in medicine, food and biotechnology. Oxford, U.K. September 13-14 2017. iii List of Abbreviations ORF Open Reading Frames CHAP Cysteine/Histidine-Dependent Amidohydrolase/Peptidase DNA Deoxyribonucleic Acid RNA Ribonucleic Acid CWBD Cell Wall Binding Domain EDTA Ethylenediaminetetraacetic Acid TMD Transmembrane Domain LAB Lactic Acid Bacteria GIT Gastrointestinal Tract PCR Polymerase Chain Reaction SPR Surface Plasmon Resonance MRSA Methicilin resistant Staphylococcu aureus BHI Brain Heart Infusion CFU Colony Forming Unit PFU Plaque Forming Unit EOP Efficiency of Plaquing MOI Multiplicity of Infection SDS Sodium Dodecyl Sulphate LTR Long Terminal Repeats NCBI National Centre for Biotechnology Information BLAST Basic Local Alignment Search Tool SDS-PAGE Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoris OD Optical Density RBP Receptor Bnding Protein iv PBS Phoshate Buffered Saline VICTOR Virus Classification and Tree Building Online Resource SOE Splicing by Overlap Extention GRAS Generally Regarded as Safe IPTG Isopropyl β-D_1-thiogalactopyranoside SLPA Surface Layer Protein A BRP Bacteriocin Release Protein LB Luria-Bertani MRS De Man, Rogosa and Sharpe MLST Multilocus Sequence Typing TEM Transmission Electron Microscopy v Table of Contents 1.1Abstract ............................................................................................................................. 1 1.2 Introduction ...................................................................................................................... 2 1.3 Structure and function of phage lysins ............................................................................. 5 1.3.1 Cell wall binding domain (CWBD) .............................................................................. 5 1.3.2 Endolysin catalytic domain ........................................................................................... 6 1.3.3 Lysin activity ................................................................................................................. 8 1.3.4 Resistance to endolysins ............................................................................................. 10 1.4 Phage-encoded proteins associated with lysins .............................................................. 10 1.4.1 Holins .......................................................................................................................... 10 1.4.2 Signal sequences ......................................................................................................... 11 1.4.3 Spanins ........................................................................................................................ 12 1.5 Protein engineering ........................................................................................................ 13 1.5.1 Domain swapping and shuffling ................................................................................. 13 1.5.2 Mutagenesis ................................................................................................................. 14 1.5.3 Lysin translocation ...................................................................................................... 15 1.6 Applications of lysins ..................................................................................................... 16 1.6.1 Food biopreservation ................................................................................................... 16 1.6.2 Lysins as therapeutics.................................................................................................. 17 1.6.3 Biofilm elimination by lysins ...................................................................................... 19 1.6.4 Diagnostic applications ..............................................................................................
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