Elucidation of the cell surface lipooligosaccharide structure of Moraxella bovoculi and its influence on the growth and biological activity of the microorganism Author Dawood, Wisam Al-Ako Published 2018 Thesis Type Thesis (Masters) School School of Medical Science DOI https://doi.org/10.25904/1912/2346 Copyright Statement The author owns the copyright in this thesis, unless stated otherwise. Downloaded from http://hdl.handle.net/10072/382710 Griffith Research Online https://research-repository.griffith.edu.au Elucidation of the cell surface lipooligosaccharide structure of Moraxella bovoculi and its influence on the growth and biological activity of the microorganism by Wisam Al-ako Dawood (BBiomedSc) School of Medical Science Griffith University, Gold Coast A dissertation submitted to the School of Medical Science, Faculty of Health Griffith University, Gold Coast in fulfilment of the requirements for the degree of Master of Medical Research October 2018 STATEMENT OF ORIGINALITY I, Wisam Dawood, hereby that this work has not previously been submitted for a degree or diploma at any university. To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made in the thesis itself. (Signed)_____________________________ Wisam Al-ako Dawood I TABLE OF CONTENTS STATEMENT OF ORIGINALITY .................................................................................... I ACKNOWLEDGEMENTS .............................................................................................. VI LIST OF FIGURES ........................................................................................................ VII LIST OF TABLES .......................................................................................................... XII LIST OF ABBREVIATIONS ........................................................................................ XIII ABSTRACT......................................................................................................................... 1 CHAPTER I: INTRODUCTION TO MORAXELLA BOVOCULI AND THE LIPOPOLYSACCHARIDE OF GRAM-NEGATIVE BACTERIA ................................. 4 1.1. Moraxella bovoculi and Infectious Bovine Keratoconjunctivitis .................................. 5 1.1.1. Historical Context ................................................................................................ 5 1.1.2. Infectious Bovine Keratoconjunctivitis and Current Causes .................................. 6 1.1.3. Current Treatments ............................................................................................... 8 1.1.4. Relationship of Moraxella bovoculi to IBK and Moraxella bovis .......................... 9 1.2. Characteristics of Gram-Negative Bacteria................................................................ 12 1.2.1. The Lipopolysaccharide Structure (LPS) ............................................................ 13 1.2.1.1. The Lipid A and Kdo ................................................................................... 14 1.2.1.2. The Core Oligosaccharide and Repeating O-Antigen ................................... 14 1.2.1.3. Lipopolysaccharide Structure in Relation to Moraxella bovis and Moraxella catarrhalis ................................................................................................................ 16 1.3. Aims, Objectives and Significance of this Research Project ...................................... 19 1.4. Research Design and Rational of Experimental Procedures ....................................... 21 1.4.1. Preliminary Genetic analysis .............................................................................. 21 1.4.2. Bacterial Growth and Activity Analysis of Moraxella bovoculi .......................... 21 1.4.3. Structural Analysis of Moraxella bovoculi .......................................................... 25 1.4.3.1. Extraction and Purification of Lipopolysaccharides ...................................... 25 1.4.3.2. SDS-PAGE Analysis.................................................................................... 26 1.4.3.3. Liquid State Nuclear Magnetic Resonance Spectroscopy.............................. 27 1.4.3.3.1. 1H NMR ................................................................................................ 28 1.4.3.3.2. 1D COSY .............................................................................................. 29 1.4.3.3.3. 1D TOCSY ............................................................................................ 29 1.4.3.3.4. 1D NOESY............................................................................................ 29 II 1.4.3.3.5. 2D 1H13C-HSQC .................................................................................... 30 1.5. Research Questions and Hypotheses ......................................................................... 31 CHAPTER II: PRELIMINARY GENETIC ANALYSIS ............................................... 33 2.1. Overview .................................................................................................................. 34 2.2. Methodology of Sequence Alignments ...................................................................... 35 2.3. Results from NCBI Blast Analysis ............................................................................ 38 2.4. Summary and Significance of Results ....................................................................... 40 CHAPTER III: MATERIALS AND METHODS ............................................................ 41 3.1. Materials ................................................................................................................... 42 3.1.1. Bacterial Strains ................................................................................................. 42 3.1.2. Reagents ............................................................................................................. 42 3.2. Methods for the Growth and Biological Activity of Moraxella bovoculi.................... 44 3.2.1. Preparation of Media .......................................................................................... 45 3.2.2. Long-Term Storage of Bacterial Cells Using Glycerol Stocks ............................. 45 3.2.3. Growth Profile Analysis of Moraxella bovoculi .................................................. 45 3.2.4. Limulus Amebocyte Lysate Assay ...................................................................... 46 3.2.5. Auto/Self-aggregation ........................................................................................ 46 3.2.6. Adherence Assay ................................................................................................ 47 3.2.7. Susceptibility to Hydrophobic Agents................................................................. 48 3.2.8. Susceptibility to Antibiotic Agents ..................................................................... 49 3.2.9. Bactericidal Assay .............................................................................................. 50 3.3. Methods for Extraction, Purification and Structural Analysis of Moraxella bovoculi . 51 3.3.1. Bacterial Culturing Methods ............................................................................... 53 3.3.2. Extraction of Lipopolysaccharides ...................................................................... 54 3.3.2.1. Phenol-Chloroform-Petroleum Ether Extraction ........................................... 54 3.3.2.2. Phenol/EDTA/TEA Extraction ..................................................................... 56 3.3.2.3. Hot Phenol/Water extraction ........................................................................ 57 3.3.3. Qualitative Analysis of Carbohydrates ................................................................ 58 3.3.4. Purification of Lipopolysaccharides from Proteins and Nucleic Acids ................ 59 3.3.5. SDS-PAGE Analysis .......................................................................................... 60 3.3.5.1. Hand-casting Polyacrylamide Gels ............................................................... 60 3.3.5.2. SDS-PAGE Protocol .................................................................................... 61 3.3.5.3. Silver-Staining of Gel .................................................................................. 61 3.3.6. Cleavage of Lipopolysaccharides Using Acid Hydrolysis ................................... 62 III 3.3.7. Size Exclusion Chromatography ......................................................................... 62 3.3.8. 1H NMR Spectroscopy and Removal of Exchangeable Protons........................... 63 3.3.9. Structural Analysis of Oligosaccharide using NMR Spectroscopy ...................... 64 CHAPTER IV: RESULTS FOR THE GROWTH AND BIOLOGICAL ACTIVITY OF MORAXELLA BOVOCULI ............................................................................................... 65 4.1. Growth Profile of Moraxella bovoculi ....................................................................... 66 4.2. Limulus Amebocyte Lysate (LAL) Assay.................................................................. 68 4.3. Auto–Aggregation Assay .......................................................................................... 71 4.4. Adherence Assay ...................................................................................................... 73 4.5. Susceptibility to Hydrophobic Agents ......................................................................