Molecular mechanisms of the human pathogen Candida glabrata involved in the interaction with the host PhD Thesis in partial fulfilment of the requirements for the degree “Doctor of Philosophy (PhD)” in the Molecular Biology Program at the Georg August University Göttingen, Faculty of Biology submitted by Pia Schmidt born in Halle/Saale, Germany Göttingen, September 2007 Affidavit Herewith I declare that my PhD thesis “Molecular mechanisms of the human pathogen Candida glabrata involved in the interaction with the host” has been written independently and with no other sources and aids than quoted. ……………………….. Pia Schmidt, Göttingen, September 30th 2007 Meiner Familie TABLE OF CONTENTS i TABLE OF CONTENTS TABLE OF CONTENTS............................................................................................................................i ACKNOWLEDGEMENTS .......................................................................................................................iv ABSTRACT................................................................................................................................................v 1 INTRODUCTION............................................................................................................................1 1.1 Fungi.......................................................................................................................................1 1.2 Medical relevance of Candida infections............................................................................2 1.3 Candida glabrata ......................................................................................................................4 1.3.1 Candida glabrata - a pathogenic yeast ..............................................................................4 1.3.2 Treatment of Candida glabrata infections.......................................................................7 1.3.3 Adaptation mechanisms ..................................................................................................9 1.3.4 Candida glabrata genetics.................................................................................................10 1.4 The fungal cell wall.............................................................................................................13 1.4.1 Cell wall structure...........................................................................................................13 1.4.2 GPI-anchored cell wall proteins ..................................................................................16 1.4.3 The GPI-anchored protein Cwp1p .............................................................................19 1.4.4 Tools for the identification of cell wall proteins .......................................................21 1.5 Aims of the study................................................................................................................22 2 MATERIALS AND METHODS ....................................................................................................25 2.1 Chemicals and Disposables...............................................................................................25 2.2 Enzymes and Reaction kits ...............................................................................................25 2.3 Synthetic oligonucleotides and molecular weight standards.........................................25 2.4 Plasmids................................................................................................................................26 2.4.1 Constructs and strategies for gene knock out............................................................27 2.4.2 Constructs and strategies for gene complementation...............................................29 2.5 Strains ...................................................................................................................................30 2.5.1 Escherichia coli strains.......................................................................................................30 2.5.2 Candida glabrata strains ...................................................................................................31 2.6 Media ....................................................................................................................................32 2.7 Software/web interfaces....................................................................................................34 2.8 Molecular Biology Methods ..............................................................................................34 2.8.1 Polymerase chain reaction (PCR).................................................................................34 2.8.2 Plasmid DNA isolation from Escherichia coli...............................................................35 2.8.3 Genomic DNA isolation from Candida glabrata.........................................................35 2.8.4 RNA isolation from Candida glabrata ...........................................................................35 2.8.5 DNA restriction enzyme digest....................................................................................36 2.8.6 Agarose Gel Electrophoresis........................................................................................36 2.8.7 DNA extraction from agarose gel ...............................................................................36 2.8.8 DNA dephosphorylation ..............................................................................................37 2.8.9 Ligation............................................................................................................................37 2.8.10 Sequencing of plasmid DNA...................................................................................37 2.8.11 Southern Blot .............................................................................................................37 2.8.11.1 DNA preparation ................................................................................................37 2.8.11.2 Blotting procedure...............................................................................................38 2.8.11.3 Preparation of a DIG-labeled probe...................................................................38 2.8.11.4 Hybridization of the DIG-labeled probe to DNA .............................................39 ii TABLE OF CONTENTS 2.8.11.5 Detection of the hybridized DIG-labeled probe................................................ 39 2.8.12 Reverse transcription................................................................................................ 39 2.8.13 Immunofluorescence microscopy .......................................................................... 40 2.8.14 Electron microscopy................................................................................................. 40 2.8.15 Time laps microscopy............................................................................................... 40 2.9 Protein biochemical methods........................................................................................... 41 2.9.1 Protein expression in Escherichia coli ............................................................................ 41 2.9.2 Protein purification from Escherichia coli..................................................................... 41 2.9.3 SDS PAGE..................................................................................................................... 42 2.9.4 Gradient SDS PAGE.................................................................................................... 43 2.9.5 Immunoblotting............................................................................................................. 43 2.9.6 Antiserum production................................................................................................... 44 2.9.7 Antiserum purification.................................................................................................. 44 2.9.8 Protein extraction from Candida glabrata .................................................................... 45 2.9.9 Candida glabrata cell wall preparation........................................................................... 46 2.9.10 Enzymatic release of GPI-anchored cell wall proteins........................................ 47 2.9.11 Chemical release of GPI-anchored cell wall proteins.......................................... 47 2.9.12 Release of mild alkali extractable cell wall proteins.............................................. 48 2.9.13 Protein quantification............................................................................................... 48 2.9.14 Two dimensional SDS PAGE................................................................................. 48 2.9.15 Preparation of the gels for image analysis ............................................................. 49 2.9.16 Image analysis............................................................................................................ 50 2.9.17 Protein preparation for mass spectrometry........................................................... 50 2.9.18 Mass spectrometric analysis of proteins ................................................................ 50 2.10 Microbiological methods................................................................................................... 51 2.10.1 Heat shock transformation of Escherichia coli........................................................