Investigating the mechanism of action of potato extract against Helicobacter pylori

12 DECLARATION.13 COPYRIGHT STATEMENT . 13 ACKNOWLEDGEMENT . 14 CHAPTER 1 . 14 INTRODUCTION . 15 1.1 Helicobacter pylori . 15 1.1.1 Genetics . 15 1.1.2 Morphology, growth and cultural characteristics. 17 1.2 Helicobacter pylori as a disease pathogen . 18 1.3 Colonisation and virulence factors . 19 1.3.1 Motility . 19 1.3.2 Urease activity . 21 1.3. 3 Outer membrane components . 22 1.3. 3.1 Outer membrane proteins . 23 1.3. 3.2 Lipopolysaccharide (LPS) outer membrane . 25 1.3. 4 Cytotoxic-associated antigen, CagA . 26 1.3. 5 Vacuolating cytotoxin, VacA . 33 1.4 Pathogenesis.36 1.5 Clinical outcome of H. pylori infection . 40 1.6 Treatment of H. pylori infection . 41 1.6. 1 Clinical treatment . 41 1.6. 2 H. pylori antibiotic resistance . 44 1.6. 3 Alternative therapies . 47 1.7 Plants as a source of antimicrobial agents . 48 1.7. 1 Mechanism of action . 51 1.7. 2 Plants exhibiting anti-H. pylori activity . 53 1.8 Potato Plant (Solanum tuberosum) . 54 1.9 Aims and Objectives . 57 CHAPTER 2 . 58 [3] MATERIALS AND METHODS . 58 2.1 Bacterial Strains, Media and Growth Conditions . 58 2.1.1 Chemicals, Reagents and Antimicrobial drugs . 58 2.1.2 Strain Maintenance and Growth conditions . 58 2.2 DNA Manipulation . 61 2.2.1 Extraction of Chromosomal DNA . 61 2.2.2 Restriction Endonuclease digestion of DNA . 61 2.2.3 Agarose Gel Electrophoresis . 61 2.2.4 Natural Transformation of H. pylori . 62 2.3 Protein Purification and Analysis . 62 2.3.1 Cellular fractionation . 62 2.3.1.1 Total membrane protein extraction . 62 2.3.1.2 Periplasmic protein extraction . 63 2.3.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) . 63 2.3.2.1 Resolving gel (12 %) . 64 2.3.2.2 Stacking gel (5 %) . 64 2.3. 3 Protein quantification . 65 2.4 Polyacrylamide gel electrophoresis of lipopolysaccharide . 65 2.4. 1 Preparation of whole-cell lysates for LPS analysis . 65 2.4. 2 TricineSDS -Polyacrylamide gel electrophoresis (T-SDS-PAGE) . 66 2.4. 2.1 T-SDS-PAGE Resolving gel . 66 2.4. 2.2 T-SDS-PAGE Stacking gel. 67 2.5 Visualisation of LPS by silver staining . 67 2.6 Morphological Test (Gram staining) . 68 2.7 Biochemical test (Urease test) . 68 2.8 Antibacterial assays . 69 2.8. 1 Viable count assay . 69 2.8. 2 Agar dilution method . 69 2.8. 3 Oxoid MIC Evaluator Method . 70 2.9 Plant Material . 70 2.10 Standardized extraction of potato extracts . 70 2.11 Determination of the protein and carbohydrate concentration in potato extract . 71 2.11.1 Determination of carbohydrate concentration . 71 2.11.2 Determination of protein concentration . 71 2.12 Preparation of concentrations of potato extracts . 72 2.13 Antibacterial activity of potato extracts . 72 2.14 Determination of minimum inhibitory concentration . 72 [4] 2.15 Bactericidal activity of potato extract (Time-kill assay) . 72 2.16 Stability of potato extracts over a specified period under different storage conditions . 73 2.17 Toxicity of potato extract towards closely-related species to H. pylori . 73 2.18 Toxicity of potato extract towards clinical antibiotic resistant strains . 74 2.19 Synergy between potato extract and antibiotics . 74 2.20 Effect of potato extract on the morphology of H. pylori . 75 2.21 Effect of potato extract on the membrane integrity of H. pylori . 76 2.22 Effect of potato extract on the membrane potential of H. pylori . 76 2.23 Effect of potato extract on the ATP concentration of H. pylori . 77 2.24 Chemical mutagenesis of H. pylori . 78 2.25 Generation H. pylori strains resistant to potato extract . 79 2.26 Statistical Analysis . 80 CHAPTER 3 . 81 EVALUATING THE ANTIBACTERIAL ACTIVITY OF POTATO EXTRACT . 81 3.