This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Bio-Oligomers as Antibacterial Agents and Strategies for Bacterial Detection Jagath C. Kasturiarachchi Ph.D. The University of Edinburgh College of Medicine & Veterinary Medicine 2014 i Declaration This thesis represents my own work. All of the experiments described herein were performed by me. I received technical assistance to perform flow cytometry and confocal analysis as declared in the acknowledgements. All chemical .entities were synthesised by the Department of Chemistry at University of Edinburgh. …………………………………………. J C Kasturiarachchi ii Acknowledgements I am very grateful to Dr Kev Dhaliwal, Professor Mark Bradley and Professor Chris Haslett for their supervision and support. I would like to thank Dr Jeff Walton and Dr Nicos Avlonitis designing and synthesis of peptoid library. I would like to thank Dr Nicos Avlonitis, Dr Marc Vendrell and Miss Alize Marangoz for their support for the synthesis of peptide probes. I wish to thank Miss Emma Scholefield for her support during my studies including killing assay development and confocal microscopy studies. My sincere thanks go to Dr Donald Davidson’s group and Professor Adriano Rossi’s group for their valuable comments and support during my PhD. I would like to thank Mrs Shonna Johnston, Mrs Fiona Rossi and Mr Will Ramsay for their technical support performing flow cytometry and confocal microscopy studies. My special thanks go to Edinburgh Global Research Scholarship, Medicine and Veterinary Medicine and Medical Research Council for their financial support during my PhD. Finally, I am eternally grateful to my parents and my brothers for their encouragement and support throughout my PhD. iii List of Figures Page Figure 1.1 Timeline of the discovery of the most important antibiotic classes. 4 Figure 1.2 Bacterial targets of the most common antibiotics. 8 Figure 1.3 Schematic representation of major resistance strategies. 12 Figure 1.4 Schematic representation of the genetics and spread of drug resistance. 14 Figure 1.5 The antimicrobial peptides discoveries each year from 1970 to 2012. 15 Figure 1.6 AMP characters and function distribution. 18 Figure 1.7 Overview of the biological activities of AMPs and Host Defence Peptides. 19 Figure 1.8 Structural features of the cell walls of Gram-positive, Gram-negative bacteria and fungus. 22 Figure 1.9 Lipid composition of phospholipids present in the cell membranes of different bacteria. 25 Figure 1.10 Mode of action for intracellular antimicrobial peptide activity. 28 Figure 1.11 Schematic representation of Polypeptoids. 31 Figure 1.12 Schematic representation of Solid-phase submonomer method. 31 Figure 1.13 The core structure of 9-mer peptoid. 39 Figure 1.14 The chemical formula of lysine amino acid and peptide to peptoid modification. 39 Figure 1.15 Schematic diagram of the development and design of the peptoid library. 40 Figure 1.16 The structure of PBP2A. 49 Figure 1.17 Aptamer library and a simplified schematic of systematic evolution of ligands by exponential enrichment (SELEX). 52 Figure 1.18 Applications of aptamer. 60 Figure 1.19 Schematic structure of a phage displaying multiple copies of a single-chain antibody. 65 Figure 2.1 Representative microscopic images of normal and apoptotic neutrophils. 83 Figure 3.1 TOP is the most antibacterial peptoid against Gram- negative bacteria. 107 Figure 3.2 TOP is the most antibacterial peptoid against Gram- positive bacteria. 108 Figure 3.3 PBS killing assay data demonstrates that TOP kills non- dividing gram-positive and gram-negative bacteria. 110 Figure 3.4 TOP shows bactericidal effect on dividing bacteria in MHB. 112 Figure 3.5 MIC for TOP against gram-positive bacteria. 114 Figure 3.6 MIC for TOP against gram-negative bacteria. 115 Figure 3.7 TOP haemolysis activity. 116 iv Figure 3.8 Schematic diagram shows the bacteria and A549 co-culture assay. 117 Figure 3.9 TOP inhibits bacterial growth in co-culture assay. 118 Figure 3.10 A549 epithelial cells are protected by TOP in co-culture assay. 119 Figure 3.11(a) Confocal microscope live image of A549 cells after incubation with TAMRA-control and TOP-TAMRA. 120 Figure 3.14(b) Kinetics of TOP-TAMRA A549 cellular uptake. 121 Figure 3.12 Schematic diagram of the internalisation assay and assessment of intracellular activity of TOP. 122 Figure 3.13 TOP demonstrates intracellular bacterial killing. 123 Figure 3.14 TOP pre-treated A549 cells reduced intracellular RN6390- gfp-lux. 124 Figure 3.15 TEM images demonstrate bacterial cell membrane disruption. 125 Figure 3.16 TOP induces ROS production in MRSA. 126 Figure 3.17 Induction of neutrophil apoptosis and necrosis by TOP at 6 hrs. 128 Figure 3.18 Induction of neutrophil apoptosis and secondary necrosis by TOP at 20 hrs. 129 Figure 3.19 QVD blocks secondary necrosis induced by TOP at 6 hrs. 130 Figure 3.20 QVD blocks secondary necrosis induced by TOP at 20 hrs 131 Figure 3.21 TOP induces rapid secondary necrosis of apoptotic neutrophils. 132 Figure 3.22 TOP has low toxicity on A549 cells. 133 Figure 3.23 TOP toxicity on A549 cells at 24 hrs incubation. 133 Figure 4.1 The map of the restriction enzyme digestion of mecA gene. 148 Figure 4.2 Agarose gel electrophoresis images; PCR amplification of mecA gene in MRSA. 149 Figure 4.3 The plasmid vector pET-21d(+). 150 Figure 4.4 Agarose gel electrophoresis image. Showing restriction digested products. 151 Figure 4.5 Agarose gel electrophoresis image showing the mecA gene amplification in 10 different colonies of BL21. 151 Figure 4.6 ClustalW data showing the alignment of sequenced data (JCK138-34-A) with the mecA gene sequence. 152 Figure 4.7 SDS-polyacrylamide protein gel showing the over expressed PBP2A protein and purified protein. 153 Figure 4.8 Latex beads agglutination assay for purified PBP2A protein. 154 Figure 5.1 Agarose (4% TBE) gel electrophoresis image showing the products of the naïve eight aptamer libraries (1ul/ lane). 160 Figure 5.2 Agarose (4% TBE) gel electrophoresis image showing the PCR amplified G and M aptamer libraries (5µl/ lane). 161 Figure 5.3 Agarose (4% TBE) gel electrophoresis image showing the PCR amplified G and M aptamer libraries after first round of SELEX. 162 v Figure 5.4 Agarose (4% TBE) gel electrophoresis image showing the products of the various PCR cycles (10, 15 and 35) of selected DNA library amplification. 163 Figure 5.5 Agarose (4% TBE) gel electrophoresis image showing the PCR products before and after ssDNA preparation. 164 Figure 5.6 Agarose (4% TBE) gel electrophoresis image showing the final PCR products before sending for Ion Torrent sequencing. 166 Figure 5.7 Electropherogram Summary from Agilent bio analyser 167 Figure 5.8 The hairpin structures of MRSA and PBP2A aptamer probes. 170 Figure 5.9 Ni-NTA protein binding assay shows that the PBP2A- aptamer is more specific to PBP2A protein compared with the BSA, FBS and Trypsin. 172 Figure 5.10 Ni-NTA protein binding assay; Cellvizio image analysis. 173 Figure 5.11 Ni-NTA protein binding assay shows that the MRSA- aptamer binds to PBP2A protein at higher rate compared to FBS and BSA. 174 Figure 5.12 Flow cytometry data showing percentage increased in mean fluorescence intensity. 175 Figure 5.13 Aptamer toxicity. 176 Figure 5.14 MRSA growth inhibition assay in MHB. 177 Figure 5.15 Optical images of mice injected with Cy5.5 conjugated aptamer library. 178 Figure 6.1 This diagram shows the experimental design; four different sets of bio-panning were carried out separately using 12- mer peptide library. 187 Figure 6.2 Phage titer of eluted phage on IPTG/Xgal Agar plate. 188 Figure 6.3 Agarose (1% TBE) gel electrophoresis image showing the phage DNA before sending for Sanger sequencing. 188 Figure 6.4 ClustalW analysed data; 1. Clones from 41 to 60; MRSA with counter selection. 190 Figure 6.5 ClustalW analysed data; 2. Clones from 61 to 80; PBP2A with counter selection. 191 Figure 6.6 ClustalW analysed data; 3. Clones from 81 to 100; MRSA no counter selection. 191 Figure 6.7 ClustalW analysed data; 4. Clones from 101to 120; PBP2A no counter selection. 192 Figure 6.8 Flow cytometry data showing the FAM-AM3 peptide binding to gram positive bacteria. 194 Figure 6.9 Flow cytometry data showing the AM3 peptide binding to gram negative bacteria. 195 Figure 6.10 The mean fluorescent intensity of the flow cytometry data. 196 Figure 6.11 Live confocal microscopic images showing the precipitation of the peptide probe AM3 in PBS incubated with MRSA. 196 vi List of Tables Page Table 1.1 Sources of clinically used classes of antibacterial drugs. 6 Table 1.2 Targets of clinically used classes of antibacterial drugs. 9 Table 1.3 Summary table of the peptoid library. 41 Table 1.4 Aptamers undergoing clinical trials. 58 Table 2.1 Oligonucleotide primers used for cloning of mecA gene in this study.