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Structural and Functional Characterization O Julius-Maximilians-Universität Würzburg Approaching antimicrobial resistance – Structural and functional characterization of the fungal transcription factor Mrr1 from Candida albicans and the bacterial ß-ketoacyl-CoA thiolase FadA5 from Mycobacterium tuberculosis Auf den Spuren der antimikrobiellen Resistenz – Strukturelle und funktionelle Charakterisierung des Transkriptionsfaktors Mrr1 aus Candida albicans und der bakteriellen β-ketoacyl-CoA thiolase FadA5 aus Mykobakterium tuberculosis Dissertation for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg, Section Biomedicine submitted by Christin Marliese Schäfer from Miltenberg am Main Würzburg, November 2014 Submitted on: ………………………………………………………..…… Office stamp Members of the Promotionskomitee: Chairperson: Ulrike Holzgrabe Primary Supervisor: Caroline Kisker Supervisor (Second): Joachim Morschhäuser Supervisor (Third): Nicole Sampson Date of Public Defence: …………………………………………….… Date of Receipt of Certificates: ……………………………………… Table of content Table of content Table of content ....................................................................................................................................... I Summary ................................................................................................................................................. 1 Zusammenfassung ................................................................................................................................... 4 1 Introduction ..................................................................................................................................... 7 1.1 The emergence of antimicrobial resistances .......................................................................... 7 1.2 Fungal infections, resistance in Candida albicans and the role of the transcription factor Mrr1 7 1.2.1 Fungal infections ............................................................................................................. 7 1.2.2 Candida species and Candida albicans ............................................................................ 8 1.2.3 Virulence factors.............................................................................................................. 9 1.2.4 Antimycotics .................................................................................................................. 10 1.2.4.1 Polyenes …………………………………………………………………………………………………………. 11 1.2.4.2 Fluoropyrimidines ………………………………………………………………………………….………. 11 1.2.4.3 Azoles ….…………………………………………………………………………………………………………. 12 1.2.4.4 Echinocandins…………………………………………………………………………………………………. 13 1.2.4.5 Others …………………………………………………………………………………………………………….. 11 1.2.5 Resistance mechanisms in C. albicans ........................................................................... 14 1.2.5.1 Structure specific and independent resistance mechanisms ……….…………………. 14 1.2.5.2 Regulation of efflux pump expression ……………………………….……….………………….. 16 1.2.6 Multidrug resistance regulator 1 (Mrr1) ...................................................................... 17 1.3 Mycobacterium tuberculosis – FadA5 involved in a new druggable pathway? ................... 21 1.3.1 Mycobacterium tuberculosis and the disease .............................................................. 21 1.3.1.1 Tuberculosis …………………………………………………………………………………………………... 21 1.3.1.2 Mycobacterium tuberculosis ……….…………………………………………………………………. 22 1.3.1.3 Infection, granuloma formation and persistence ……………………………………………. 23 1.3.2 Treatment of tuberculosis, anti-TB drugs and resistance ............................................. 24 1.3.3 Approaching new ways – The cholesterol metabolism pathway .................................. 27 1.3.4 The β-ketoacyl-CoA thiolase FadA5 ............................................................................... 30 1.3.4.1 Role in M. tuberculosis infection ……………………………………………………………………. 30 1.3.4.2 Thiolase activity and reaction of FadA5 ………………………………………………………….. 32 1.3.4.3 What about human thiolase homologs? ………………………………………………………... 33 1.4 Research objective ................................................................................................................ 34 2 Materials ........................................................................................................................................ 35 2.1 Chemicals ............................................................................................................................... 35 I Table of content 2.2 Equipment ............................................................................................................................. 35 2.3 Enzymes, Kits and Additives .................................................................................................. 37 2.4 Bacteria and Yeast culture ..................................................................................................... 38 2.4.1 Bacterial strains, yeast strains and plasmids ................................................................. 38 2.4.2 Agarose gel electrophoresis .......................................................................................... 40 2.4.3 Antibiotics, media and additives ................................................................................... 40 2.5 Protein purification ............................................................................................................... 41 2.5.1 Buffers and solutions ..................................................................................................... 41 2.5.2 Protein gel electrophoresis ........................................................................................... 43 2.5.3 Western blotting ........................................................................................................... 43 2.6 Crystallization screens and compounds ................................................................................ 43 2.7 Software and Databases ........................................................................................................ 45 3 Methods ........................................................................................................................................ 46 3.1 Molecular biology and microbiology techniques .................................................................. 46 3.1.1 Polymerase chain reaction (PCR) .................................................................................. 46 3.1.2 DNA agarose gel electrophoresis .................................................................................. 47 3.1.3 DNA purification ............................................................................................................ 47 3.1.4 Transformation and genetic integration ....................................................................... 48 3.1.4.1 Transformation into E. coli cells ….……….………………………………………………………... 48 3.1.4.2 Transformation into M. smegmatis cells – electroporation ……………………………. 49 3.1.4.3 Genetic integration into P. pastoris …………………………………………………….…………. 50 3.1.5 Plasmid amplification and isolation............................................................................... 51 3.1.6 Cloning Techniques ....................................................................................................... 52 3.1.6.1 Restriction digestion and ligation ….……….……………………………………………………... 52 3.1.6.2 Sequence and ligantion independent cloning (SLIC) …………………………………….... 54 3.1.6.3 Site-directed mutagenesis …………….……….………………………………………………………. 55 3.1.6.4 Colony PCR – E. coli ….…………………………….……………………………………………………... 56 3.1.6.5 Colony PCR – P. pastoris ……..………………….……………………………………………………... 56 3.1.7 Protein expression ......................................................................................................... 57 3.1.7.1 E. coli …………………………………….…..…………………………………………………………………... 57 3.1.7.2 M. smegmatis …………………………….…..…………………………………………………………..... 57 3.1.7.3 P. pastoris ……….…………….…..………………….…………………………………………………….... 57 3.2 Protein purification and characterization ............................................................................. 59 3.2.1 Protein purification........................................................................................................ 59 3.2.1.1 Cell lysis ……………………….…..………………………………………………………………………….... 60 II Table of content 3.2.1.1.1 FadA5 from M. smegmatis …………………………………………………………………. 60 3.2.1.1.2 Mrr1 variants from E. coli ………………………………………………..…………………. 60 3.2.1.1.3 Mrr1 variants in P. pastoris ………………………………………………..………………. 61 3.2.1.2 Metal-affinity chromatography ……..…………………………………………………………….... 61 3.2.1.3 Anion-exchange chromatography ……..………………………………………………………..... 63 3.2.1.4 TEV-cleavage reaction ………………….…..………………………………………………………...... 64 3.2.1.5 Size-exclusion chromatography ……..…………………………………………………………....... 65 3.2.2 Protein characterization ................................................................................................ 66 3.2.2.1 SDS-PAGE gel electrophoresis …..…..…………………………………………………………....... 66 3.2.2.2 Western Blotting ……………………...…..…………………………………………………………....... 66 3.2.2.3 Thermofluor assay …………………...…..…………………………………………………………....... 67 3.2.2.4 Circular-dichroism (CD) spectroscopy ………………………………………………………....... 68 3.2.2.5 Dynamic light scattering (DLS) ….…..…………………………………………………………....... 69 3.2.2.6 Size-exclusion chromatography coupled multiangle laser light scattering (SEC-MALLS) 69 3.2.2.7 Isothermal
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