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The Cellular and Molecular Responses of Aspergillus Fumigatus to the Antifungal Drug Caspofungin

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The Cellular and Molecular Responses of Aspergillus Fumigatus to the Antifungal Drug Caspofungin The cellular and molecular responses of Aspergillus fumigatus to the antifungal drug caspofungin A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Sergio David Moreno Velásquez School of Biological Sciences Infection, Immunity and Respiratory Medicine Manchester Fungal Infection Group CONTENT Page Title…………………………………………………………………………………. 1 Contents……………………………………………………………………………... 2 List of Figures………………………………………………………......................... 8 List of Tables………………………………………………………………………... 11 Abbreviations…………………………………………………………...................... 13 Abstract……………………………………………………………………………... 17 Declaration………………………………………………………………………….. 19 Copyright statement………………………………………………............................ 19 Acknowledgements………………………………………………………................. 20 Overview of Thesis………………………………………………………………… 21 Chapter 1………………………………………………………………………….... 25 1. Introduction………………………………………………………………………. 25 1.1. The fifth kingdom………………………………………………………………. 25 1.2. Fungal invaders……………………………………………................................ 27 1.3. Fungal infections in humans………..................................................................... 29 1.4. Aspergillus……………………………………………………………………… 33 1.5. The opportunistic fungus Aspergillus fumigatus……………………………….. 35 1.6. The cell wall of Aspergillus fumigatus…………………………………………. 41 1.7. Antifungal drugs………………………………………………………………... 50 1.8. The echinocandin caspofungin…………………………………………………. 58 2 1.9. The paradoxical effect of caspofungin…………………………………………. 62 2. Aims of the research described in this thesis…………………………………... 67 3. References……………………………………………………………………… 68 Chapter 2…………………………………………………………………………… 101 2. Materials and Methods…………………………………………………………… 101 2.1. Materials………………………………………………………………………... 101 2.1.2. Primers………………………………………………………………………... 103 2.1.3. Organisms and plasmids……………………………………………………… 107 2.2. Methods………………………………………………………………………… 108 2.2.1. Growth and maintenance of Aspergillus fumigatus………………………….. 108 2.2.2. Growth and maintenance of Escherichia coli………………………………... 111 2.2.3. Growth and maintenance of the alveolar epithelial cell line, A549 …………. 113 2.3. Bioinformatics………………………………………………………………….. 114 2.3.1. BLAST search analysis………………………………………………………. 114 2.4. Molecular methods……………………………………………………………... 114 2.4.1. Plasmid DNA preparations from Escherichia coli…………………………… 114 2.4.2. Genomic DNA extraction from Aspergillus fumigatus ……………………… 116 2.4.3. DNA precipitation……………………………………………………………. 118 2.4.4. Isolation of total RNA of Aspergillus fumigatus……………………………... 119 2.4.5. Gel electrophoresis…………………………………………………………… 120 2.5. Polymerase chain reaction (PCR)………………………………………………. 122 2.5.1. Standard polymerase chain reaction………………………………………….. 122 3 2.5.2. Touch-down PCR (TD-PCR)………………………………………………… 123 2.5.3. Fusion PCR………………………………………………………………….. 125 2.5.4. Quantitative gene expression by real-time PCR……………………………… 127 2.5.5. PCR from Aspergillus fumigatus spores…………………………………….. 128 2.6. Transformation…………………………………………………………………. 129 2.6.1. Transformation of Escherichia coli………………………………………….. 129 2.6.2. Aspergillus fumigatus transformation………………………………………... 130 2.7. Single and homologous integration into the genome of Aspergillus fumigatus... 132 2.7.1. Homologous integration screening…………………………………………… 132 2.7.2. Southern blot (DNA-DNA hybridization)……………………………………. 133 2.8. Microscopy……………………………………………………………………... 135 2.8.1. Confocal laser-scanning microscopy (CLSM)……………………………….. 135 2.8.2. Immunostaining of α-1,3-glucans……………………………………………. 135 2.9. Clinical approaches…………………………………………………………….. 136 2.9.1. Antifungal susceptibility testing……………………………………………… 136 2.9.2. Aspergillus fumigatus infection of alveolar cells…………………………….. 136 2.10. Statistical analyses…………………………………………………………….. 137 2.11. References…………………………………………………………………….. 137 Chapter 3…………………………………………………………………………… 142 3. Caspofungin-mediated growth inhibition and paradoxical growth in Aspergillus 142 fumigatus involves fungicidal hyphal tip lysis coupled with regenerative intrahyphal growth and dynamic changes in β-1,3-glucan synthase localization. 3.1. Abstract………………………………………………………………………… 142 4 3.2. Introduction…………………………………………………………………….. 143 3.3. Materials and methods………………………………………………………….. 145 3.3.1. Strains, media and growth conditions………………………………………. 145 3.3.2. Construction of Aspergillus fumigatus fks1-gfp, rho1-gfp and rfp-rab5 146 expression strains…………………………………........................................... 3.3.3. Confocal microscopy………………………………………………................. 147 3.3.4. Human A549 epithelial cell line infection assay……………………………... 148 3.3.5. Statistical analyses…………………………………………………................. 148 3.4. Results………………………………………………………………………….. 149 3.4.1. The switch to paradoxical growth in leading hyphae between 38 and 48 h 149 after continuous exposure to a high caspofungin concentration……………. 3.4.2. Caspofungin causes tip lysis followed by regenerative, intrahyphal growth.. 154 3.4.3. Caspofungin-mediated paradoxical growth response occurs during the 157 infection of human A549 alveolar epithelial cells…………………………... 3.4.4. The normal hyphal tip localization of Fks1 was switched to vacuole 160 following caspofungin treatment but restored to hyphal tips during paradoxical growth………………………………………………………….. 3.4.5. Fks1 co-localizes with the endocytic marker, Rab5, in vacuoles upon 166 caspofungin treatment………………………………………………………. 3.4.6. Localization of the regulatory subunit of the glucan synthase, Rho1, is 169 unaffected by caspofungin treatment but hyphal tip-localization of Rho1 is essential for paradoxical growth…………………………………………….. 3.4.7. Farnesol blocks paradoxical growth and the relocalization of Fks1 and 171 Rho1 to hyphal tips at high concentrations of caspofungin………………… 3.5. Discussion……………………………………………………………………… 174 5 3.6. References……………………………………………………………………… 180 Chapter 4…………………………………………………………………………… 189 4. Live-cell imaging of the dynamics of caspofungin-induced paradoxical growth 189 in the human pathogen, Aspergillus fumigatus………………………………… 4.1. Abstract………………………………………………………………………… 189 4.2. Introduction…………………………………………………………………….. 190 4.3. Materials and methods …………………………………………………………. 191 4.4. Results and discussion………………………………………………………….. 192 4.5. References……………………………………………………………………… 203 Chapter 5…………………………………………………………………………… 207 5. Caspofungin treatment induces the recruitment of the transcription factor 207 CrzA to nuclei and the reorganization of the main cell-wall components during paradoxical growth in Aspergillus fumigatus…………………………... 5.1. Abstract………………………………………………………………………… 207 5.2. Introduction…………………………………………………………………….. 208 5.3. Materials and methods…………………………………………………………. 210 5.3.1. Strains, media and growth conditions………………………………………... 210 5.3.2. Antifungal susceptibility……………………………………………………... 211 5.3.3. α-(1,3)-glucan staining in cell wall…………………………………………... 211 5.3.4. Confocal microscopy…………………………………………………………. 212 5.3.5. Measurements of different cell wall components…………………………….. 212 5.4. Results………………………………………………………………………….. 213 5.4.1. Minimal effective concentration of caspofungin……………………………... 213 6 5.4.2. Caspofungin induces the mislocalization of Fks1 in young germlings and 214 mature hyphae………………………………………………………………. 5.4.3. The mislocalization of Fks1 caused by caspofungin blocks the tip 217 production of β-1,3 glucan but the chitin production increases in mature hyphae of A. fumigatus……………………………………………………… 5.4.4. Caspofungin induces the nuclear translocation of the transcription factor, 221 CrzA………………………………………………………………………… 5.4.5. The nuclear dynamics of A. fumigatus is affected by caspofungin treatment 224 5.4.6. The vacuolar dynamics of A. fumigatus is unaffected by caspofungin……... 226 5.4.7. Colonies of A. fumigatus CEA10 exhibit paradoxical effect by 48 h………. 228 5.4.8. Some clinical azole-resistant strains show paradoxical growth…………….. 231 5.4.9. Paradoxical growth in leading hyphae is associated with normalized content of α-1,3- and β-1,3-glucan, but not chitin…………………………... 5.4.10. Paradoxical growth in A. fumigatus is not caused by caspofungin inactivity. 238 5.5. Discussion……………………………………………………………………… 240 5.6. References……………………………………………………………………… 244 Chapter 6…………………………………………………………………………… 252 6. Conclusion and future work……………………………………………………… 252 6.1. Chapter 3……………………………………………………………………….. 252 6.2. Chapter 4……………………………………………………………………….. 256 6.3. Chapter 5……………………………………………………………………….. 258 6.4. Model of the dynamics of the β-1,3-glucan synthase complex of A. fumigatus 260 hypha in response to caspofungin treatment…………….................................... 6.5. References……………………………………………………………………… 263 Word count: 55, 625 7 LIST OF FIGURES Figure Page 1.1 The mould Aspergillus fumigatus 36 1.2 Life cycle of Aspergillus fumigatus 38 1.3 Model of invasive aspergillosis development in immunocompromised 40 individuals 1.4 Comparison of the cell wall components of A. fumigatus conidium 44 and hypha 1.5 Mode of action of the most commonly used antifungal drugs: 53 polyenes, azoles and echinocandins 1.6 Chemical structures of the licenced echinocandins for the treatment 57 of invasive fungal infections 1.7 Quadriphasic nature of the paradoxical effect of Candida and 63 Aspergillus species 2.1 Gene tagging, gene replacement and promoter replacement by fusion 126 PCR products 3.1 Induction of paradoxical growth in microcolonies of A. fumigatus 151 occurs by 48 h of caspofungin treatment 3.2 Cellular development
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