Table of Contents Declaration of Authorship x Acknowledgements xi Presentations xii Abbreviations xiv Summary xviii 1.1. General characteristics of Aspergillus fumigatus. .................................................................... 1 1.2. Pathobiology of A. fumigatus. .................................................................................................. 3 1.3. Anti-fungal strategies currently in place for the treatment of IA. ............................................ 6 1.4. A. fumigatus: toxins and virulence factors. ............................................................................ 10 1.5. A. fumigatus genetics and genome information..................................................................... 15 1.6. Secondary metabolites and clustering of biosynthetic genes. ................................................ 18 1.7. Regulation of secondary metabolism gene clusters by the global secondary metabolite regulator LaeA............................................................................................................................... 20 1.8. Association of secondary metabolism with development. ..................................................... 21 1.9. Non-ribosomal peptide synthesis (NRPS).............................................................................. 24 1.9.1. NRPS – An overview ...................................................................................................... 24 1.9.2. Structural arrangements and domain architecture of NRP synthetases........................... 28 1.9.3. 4’phosphopantetheinyl transferases and requirement for NRP Synthetase activation to holoenzyme................................................................................................................................ 32 1.9.4. NRPS – Mechanisms of Non-ribosomal Peptide Synthesis. ........................................... 34 1.10. NRPS in A. fumigatus........................................................................................................... 39 1.11. Genetic manipulations available for characterisation of genes within A. fumigatus............ 41 1.11.1. Transformation systems................................................................................................. 41 1.11.2. Preparation of transforming DNA for targeted gene deletions...................................... 42 1.11.3. Selection markers used for A. fumigatus genetic manipulation..................................... 46 1.11.4. The use of non-homologous end joining (NHEJ)-deficient strains to increase targeted integrations. ............................................................................................................................... 49 1.12. The oxidative stress response in A. fumigatus...................................................................... 52 1.13. Use of the Galleria mellonella insect model to study A. fumigatus virulence..................... 55 1.14 Rationale and objectives of this thesis................................................................................... 57 ii 2.1. Materials ................................................................................................................................. 59 2.1.1 Microbiological Medias and Reagents ............................................................................. 59 2.1.1.1 Aspergillus Trace Elements....................................................................................... 59 2.1.1.2 Aspergillus Salt Solution ........................................................................................... 59 2.1.1.3 100x Ammonium Tartrate ......................................................................................... 59 2.1.1.4 20 mM L-Glutamine.................................................................................................. 59 2.1.1.5 Malt Extract Agar ...................................................................................................... 60 2.1.1.6 Aspergillus Minimal Media (AMM) Agar ................................................................ 60 2.1.1.7 Aspergillus Minimal Media (AMM) ......................................................................... 60 2.1.1.8 Yeast Glucose (YG) Media ....................................................................................... 60 2.1.1.9 Sabourard- Dextrose Media....................................................................................... 61 2.1.1.10 RPMI Media ............................................................................................................ 61 2.1.1.11 Czapek’s Broth ........................................................................................................ 61 2.1.1.12 Czapek’s Agar ......................................................................................................... 61 2.1.1.13 MEM Media supplemented with 5 % (w/v) Foetal Calf Serum (FCS)................... 61 2.1.1.14 Aspergillus Transformation Regeneration Media.................................................... 62 2.1.1.15 Aspergillus Transformation Soft Agar .................................................................... 62 2.1.1.16 Phosphate Buffered Saline (PBS)............................................................................ 62 2.1.1.17 Phosphate Buffer Saline/ 0.1 % (w/v) Tween-20 (PBST) NaCl (0.9 % w/v) ......... 63 2.1.1.18 Luria-Bertani (LB) Agar.......................................................................................... 63 2.1.1.19 Luria-Bertani (LB) Broth ........................................................................................ 63 2.1.1.20 80 % (w/v) Glycerol ................................................................................................ 63 2.1.1.21 40 % (w/v) Glycerol ................................................................................................ 63 2.1.1.22 YPD broth................................................................................................................ 64 2.1.1.23 YPD Agar ................................................................................................................ 64 2.1.1.24 Dropout Mix ............................................................................................................ 64 2.1.1.25 Synthetic Complete Media (SC).............................................................................. 64 2.1.1.26 Synthetic Complete Agar (SC)................................................................................ 64 2.1.1.27 Antibiotics and Plate Assay Supplements ............................................................... 65 2.1.2 Molecular Biological Reagents ........................................................................................ 69 2.1.2.1 Agarose Gel Electrophoresis Reagents ..................................................................... 69 2.1.2.1.1 50 X Tris-Acetate (TAE).................................................................................... 69 2.1.2.1.2 1 X Tris-Acetate (TAE)...................................................................................... 69 2.1.2.1.3 Ethidium Bromide Solution................................................................................ 69 2.1.2.1.4 SYBR® Safe DNA Gel Stain............................................................................. 69 2.1.2.1.5 1 % (w/v) Agarose Gel ....................................................................................... 69 2.1.2.2 DNA Reagents........................................................................................................... 70 2.1.2.2.1 100 % (v/v) Ethanol (ice-cold) ........................................................................... 70 2.1.2.2.2 70 % (v/v) Ethanol (ice-cold) ............................................................................. 70 2.1.2.2.3 3 M Na-acetate ................................................................................................... 70 2.1.3 Aspergillus Transformation Buffers ................................................................................. 74 2.1.3.1 Lysis Buffer ............................................................................................................... 74 2.1.3.2 Mycelial lysing solution ............................................................................................ 74 2.1.3.3 0.7 M KCl.................................................................................................................. 74 2.1.3.4 L6 Buffer ................................................................................................................... 74 2.1.3.5 L7 Buffer ................................................................................................................... 75 2.1.4 Southern and Northern Blotting Reagents........................................................................ 75 iii 2.1.4.1 Southern Transfer Buffer........................................................................................... 75 2.1.4.2 20 X SSC Buffer........................................................................................................ 75 2.1.4.3 10 X SSC Buffer........................................................................................................ 75 2.1.4.4 2 X SSC Buffer.......................................................................................................... 75 2.1.4.5 10 % (w/v) Sodium Dodecyl Sulphate (SDS) ........................................................... 76 2.1.4.6