List of Contributors xv

1 Diagnosis of Candida Infection in Tissue by Immunohistochemistry 1
Malcolm D. Richardson, Riina Rautemaa and Jarkko Hietanen

1.1 Introduction 1

1.2 Specificity of monoclonal antibody 3H8 for C. albicans 3

Protocol 1.1 Testing of specificity of monoclonal antibody 3H8 4

1.3 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6

Protocol 1.2 Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms 6

1.4 Application of immunohistochemistry in the diagnosis of Candida periodontal disease 7

Protocol 1.3 Use of monoclonal antibody 3H8 in the detection of C. albicans in tissue 8

1.5 References 11

2 Transmission Electron Microscopy of Pathogenic Fungi 13
Guy Tronchin and Jean-Philippe Bouchara

2.1 Introduction 13

2.2 Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis 16

Protocol 2.1 Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis 17

2.3 Identification of the different compartments of the secretory pathway in yeasts 18

Protocol 2.2 Identification of the different compartments of the secretory pathway in yeasts 19

2.4 Cytochemical localization of acid phosphatase in yeasts 20

Protocol 2.3 Cytochemical localization of acid phosphatase in yeasts 21

2.5 Detection of anionic sites 23

Protocol 2.4 Detection of anionic sites 23

2.6 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 25

Protocol 2.5 Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg) 26

2.7 Enzyme-gold approach for the detection of polysaccharides in the cell wall 28

Protocol 2.6 Enzyme-gold approach for the detection of polysaccharides in the cell wall 29

2.8 Detection of glycoconjugates by the lectin-gold technique 30

Protocol 2.7 Detection of glycoconjugates by the lectin-gold technique 31

2.9 Immunogold detection of antigens on ultrathin sections of acrylic resin 33

Protocol 2.8 Immunogold detection of antigens on ultrathin sections of acrylic resin 34

2.10 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 36

Protocol 2.9 Cryofixation and freeze substitution for ultrastructural analysis and immunodetection 37

2.11 Overview 38

2.12 References 39

3 Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi 43
Maria Simitsopoulou and Emmanuel Roilides

3.1 Introduction 43

3.2 Preparation of conidia and hyphae of opportunistic fungi 45

Protocol 3.1 Preparation of conidia and hyphae of opportunistic fungi 45

Protocol 3.2 Preparation of hyphal fragments 47

3.3 Isolation of human monocytes from whole blood 48

Protocol 3.3 Isolation of human MNCs from whole blood 48

3.4 Analysis of human MNC function in response to fungal infection 51

Protocol 3.4 XTT microassay 52

Protocol 3.5 Superoxide anion assay in 96-well plate 53

Protocol 3.6 Hydrogen peroxide-rhodamine assay 55

Protocol 3.7 Phagocytosis assay 55

3.5 Evaluation of immunomodulators in response to fungal infection 57

Protocol 3.8 Analysis of gene expression by RT-PCR 58

Protocol 3.9 Analysis of pathway-specific gene expression by microarray technology 63

Protocol 3.10 Assessment of cytokines and chemokines by ELISA 66

3.6 Overview 67

3.7 References 68

4 Determination of the Virulence Factors of Candida Albicans and Related Yeast Species 69
Khaled H. Abu-Elteen and Mawieh Hamad

4.1 Introduction 69

4.2 Measurement of Candida species adhesion in vitro 70

Protocol 4.1 The visual assessment of candidal adhesion to BECs 70

Protocol 4.2 The radiometric measurement of candidal adhesion 73

4.3 Adhesion to inanimate surfaces 75

Protocol 4.3 Assessment of candidal adhesion to denture acrylic material 75

Protocol 4.4 Adherence of Candida to plastic catheter surfaces 76

4.4 C. albicans strain differentiation 77

Protocol 4.5 Resistogram typing 77

Protocol 4.6 The slide agglutination technique 79

Protocol 4.7 Serotyping of C. albicans by flow cytomerty 80

4.5 Phenotypic switching in C. albicans 81

Protocol 4.8 Evaluation of phenotype switching in C. albicans 82

4.6 Extracellular enzymes secreted by C. albicans 83

Protocol 4.9 Measurement of extracellular proteinase production by C. albicans 85

Protocol 4.10 Measurement of extracellular proteinase produced by C. albicans (staib method) 86

Protocol 4.11 Measurement of extracellular phospholipases of C. albicans 87

4.7 Germ-tube formation in C. albicans 88

Protocol 4.12 Germ-tube formation assay 88

4.8 References 89

5 Analysis of Drug Resistance in Pathogenic Fungi 93
Gary P. Moran, Emmanuelle Pinjon, David C. Coleman and Derek J. Sullivan

5.1 Introduction 93

5.2 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 96

Protocol 5.1 Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts 97

5.3 Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters 102

Protocol 5.2 Measurement of rhodamine 6G uptake and glucose-induced efflux 102

5.4 Analysis of expression of multidrug transporters in pathogenic fungi 105

5.5 Analysis of point mutations in genes encoding cytochrome P- 450 lanosterol demethylase 106

5.6 Qualitative detection of alterations in membrane sterol contents 108

Protocol 5.3 Qualitative detection of alterations in membrane sterol contents 109

5.7 Overview 110

5.8 References 110

6 Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi 115
Eric Dannaoui

6.1 Introduction 115

6.2 Disseminated zygomycosis in non-immunosuppressed mice 118

Protocol 6.1 Disseminated zygomycosis in non-immunosuppressed mice 118

6.3 Animal model of disseminated aspergillosis 125

Protocol 6.2 Disseminated aspergillosis in neutropoenic mice 126

6.4 Study design for evaluation of antifungal combinations therapy in animal models 130

Protocol 6.3 Study design for the evaluation of combination therapy in animal models 131

6.5 References 133

7 Proteomic Analysis of Pathogenic Fungi 137
Alan Murphy

7.1 Introduction 137

Protocol 7.1 2D SDS-PAGE of protein samples 139

7.2 Protein digestion in preparation for mass spectrometry by MALDI-TOF 140

Protocol 7.2 Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry 141

Protocol 7.2a In-gel digestion 142

Protocol 7.2b In-solution digestion 143

7.3 MALDI-TOF mass spectrometry 145

Protocol 7.3 Preparation of matrix for MALDI-TOF 147

7.4 Peptide mass fingerprinting (PMF) 149

Protocol 7.4 Post-source decay (PSD) and chemically assisted fragmentation (CAF) 150

7.5 Interpreting MALDI-TOF result spectra 152

7.6 Overview 156

7.7 References 157

8 Extraction and Detection of DNA and RNA from Yeast 159
Patrick Geraghty and Kevin Kavanagh

8.1 Introduction 159

8.2 The extraction of yeast DNA with the aid of phenol: chloroform 161

Protocol 8.1 Whole-cell DNA extraction from C. albicans using phenol: chloroform 161

Protocol 8.2 Rapid extraction of DNA from C. albicans colonies for PCR 164

8.3 Detection of yeast DNA using radio-labelled probes 165

Protocol 8.3 DNA detection by Southern blotting 165

8.4 Extraction of whole-cell RNA using two different protocols 169

Protocol 8.4 The extraction of whole-cell RNA from yeast using phenol: chloroform 170

Protocol 8.5 Rapid extraction of whole-yeast-cell RNA 172

8.5 Detection and expression levels of specific genes by the examination of mRNA in yeast 174

Protocol 8.6 Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis 175

Protocol 8.7 Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis 176

8.6 References 179

9 Microarrays for Studying Pathogenicity in Candida Albicans 181
André Nantel, Tracey Rigby, Hervé Hogues and Malcolm Whiteway

9.1 Introduction 181

9.2 DNA microarrays 182

9.3 Building a second-generation 2-colour long oligonucleotide microarray for C. albicans 183

Protocol 9.1 Isolation of C. albicans RNA 185

Protocol 9.2 Isolation of total RNA using the hot phenol method 186

Protocol 9.3 Isolation of Poly-A+ mRNA 187

Protocol 9.4 Determination of the efficiency of incorporation of the probe 192

Protocol 9.5 Hybridization to DNA microarrays 194

9.4 Experiment design 196

9.5 Microarray-based studies in C. albicans 200

9.6 Conclusion 205

9.7 References 205

10 Molecular Techniques for Application with Aspergillus Fumigatus 211
Nir Osherov and Jacob Romano

10.1 Introduction 211

10.2 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 212

Protocol 10.1 Preparation of knockout vectors for gene disruption and deletion in A. fumigatus 213

10.3 Transformation of A. fumigatus 217

Protocol 10.2 Chemical transformation of A. fumigatus 218

10.4 Molecular verification of correct single integration (PCR-based) 220

Protocol 10.3 Molecular verification of correct integration by PCR 221

10.5 General strategies for the phenotypic characterization of A. fumigatus mutant strains 223

Protocol 10.4 General strategies for the phenotypic characterization of mutants 223

10.6 References 229

11 Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus 231
Matthias Brock, Alexander Gehrke, Venelina Sugareva and Axel A. Brakhage

11.1 Introduction 231

11.2 Site-directed mutagenesis of promoter elements 233

Protocol 11.1 Site-directed mutation of promoter elements 233

11.3 lacZ as a reporter gene 236

Protocol 11.2 lacZ as a reporter gene: discontinuous determination of b-galactosidase activity 237

Protocol 11.3 lacZ as a reporter gene: continuous determination of b-galactosidase activity 239

11.4 Transformation of A. fumigatus 241

Protocol 11.4 Transformation of A. fumigatus 242

11.5 Hygromycin B as a selection marker for transformation 246

Protocol 11.5 Hygromycin B as a selection marker for transformation 247

11.6 pyrG as a selection marker for transformation 249

Protocol 11.6 pyrG as a selection marker for transformation 249

11.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 251

Protocol 11.7 URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions 253

11.8 References 255

12 Microarray Technology for Studying the Virulence of Aspergillus Fumigatus 257
Darius Armstrong-James and Thomas Rogers

12.1 Introduction 257

12.2 Isolation of RNA from A. fumigatus 259

Protocol 12.1 Isolation of total RNA from A. fumigatus 260

12.3 Reverse transcription of RNA and fluorescent labelling of cDNA 263

Protocol 12.2 Indirect labelling of cDNA with fluorescent dyes 264

12.4 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 266

Protocol 12.3 Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing 267

12.5 Image acquisition from hybridized microarrays 269

Protocol 12.4 Image acquisition from hybridized microarrays 269

12.6 Microarray image analysis 270

Protocol 12.5 Microarray image analysis 271

12.7 References 272

13 Techniques and Strategies for Studying Virulence Factors in Cryptococcus Neoformans 275
Nancy Lee and Guilhem Janbon

13.1 Introduction 275

13.2 Construction of C. neoformans gene-disruption cassettes 278

Protocol 13.1 Construction of C. neoformans gene-disruption cassettes 278

13.3 Genetic transformation of C. neoformans 283

Protocol 13.2 Biolistic transformation of C. neoformans 283

Protocol 13.3 Transformation via electroporation 286

13.4 Extraction of genomic DNA from C. neoformans 287

Protocol 13.4 DNA for use in library construction and hybridization analysis 288

Protocol 13.5 DNA for use in PCR 291

13.5 Screening and identification of deletion strains 292

Protocol 13.6 Screening and identification of deletion strains 292

13.6 Measuring capsule size in C. neoformans 297

Protocol 13.7 Measuring capsule size in C. neoformans 298

13.7 Purification of glucuronoxylomannan (GXM) 298

Protocol 13.8 Purification of glucuronoxylomannan (GXM) 299

13.8 References 301

14 Genetic Manipulation of Zygomycetes 305
Ashraf S. Ibrahim and Christopher D. Skory

14.1 Introduction 305

14.2 Genetic tools to manipulate mucorales 306

14.3 Selectable markers used with mucorales fungi 307

14.4 Introduction of DNA used for transformation 308

Protocol 14.1 Protoplasting of R. oryzae 309

Protocol 14.2 Biolistic delivery system transformation of R. oryzae 313

Protocol 14.3 A. tumefaciens-mediated transformation 316

14.5 Molecular analysis of transformants 319

14.6 Overview 322

14.7 References 323

Index 327