Medical Mycology

Preface xiii 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