Medical Mycology: Cellular and Molecular Techniques Book

Preface     xiii
List of Contributors     xv
Diagnosis of Candida Infection in Tissue by Immunohistochemistry   Malcolm D. Richardson   Riina Rautemaa   Jarkko Hietanen     1
Introduction     1
Specificity of monoclonal antibody 3H8 for C. albicans     3
Testing of specificity of monoclonal antibody 3H8     4
Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms     6
Evaluation of monoclonal antibody 3H8 for the detection of C. albicans morphological forms     6
Application of immunohistochemistry in the diagnosis of Candida periodontal disease     7
Use of monoclonal antibody 3H8 in the detection of C. Albicans in tissue     8
References     11
Transmission Electron Microscopy of Pathogenic Fungi   Guy Tronchin   Jean-Philippe Bouchara     13
Introduction     13
Glutaraldehyde-potassium-permanganate or glutaraldehyde-osmiumtetroxide fixation for ultrastructural analysis     16
Glutaraldehyde-osmium tetroxide (#) or glutaraldehyde-potassium permanganate (*) fixation for ultrastructural analysis     17
Identification of the different compartments of the secretory pathway in yeasts     18
Identification of the different compartments of thesecretory pathway in yeasts     19
Cytochemical localization of acid phosphatase in yeasts     20
Cytochemical localization of acid phosphatase in yeasts     21
Detection of anionic sites     23
Detection of anionic sites     23
Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg)     25
Detection of glycoconjugates by the periodic acid-thiocarbohydrazide-silver proteinate technique (PATAg)     26
Enzyme-gold approach for the detection of polysaccharides in the cell wall     28
Enzyme-gold approach for the detection of polysaccharides in the cell wall     29
Detection of glycoconjugates by the lectin-gold technique     30
Detection of glycoconjugates by the lectin-gold technique     31
Immunogold detection of antigens on ultrathin sections of acrylic resin     33
Immunogold detection of antigens on ultrathin sections of acrylic resin     34
Cryofixation and freeze substitution for ultrastructural analysis and immunodetection     36
Cryofixation and freeze substitution for ultrastructural analysis and immunodetection     37
Overview     38
References     39
Evaluation of Molecular Responses and Antifungal Activity of Phagocytes to Opportunistic Fungi   Maria Simitsopoulou   Emmanuel Roilides     43
Introduction     43
Preparation of conidia and hyphae of opportunistic fungi     45
Preparation of conidia and hyphae of opportunistic fungi     45
Preparation of hyphal fragments     47
Isolation of human monocytes from whole blood     48
Isolation of human MNCs from whole blood     48
Analysis of human MNC function in response to fungal infection     51
XTT microassay     52
Superoxide anion assay in 96-well plate     53
Hydrogen peroxide-rhodamine assay     55
Phagocytosis assay     55
Evaluation of immunomodulators in response to fungal infection     57
Analysis of gene expression by RT-PCR     58
Analysis of pathway-specific gene expression by microarray technology     63
Assessment of cytokines and chemokines by ELISA     66
Overview     67
References     68
Determination of the Virulence Factors of Candida Albicans and Related Yeast Species   Khaled H. Abu-Elteen   Mawieh Hamad     69
Introduction     69
Measurement of Candida species adhesion in vitro     70
The visual assessment of candidal adhesion to BECs      70
The radiometric measurement of candidal adhesion     73
Adhesion to inanimate surfaces     75
Assessment of candidal adhesion to denture acrylic material     75
Adherence of Candida to plastic catheter surfaces     76
C. albicans strain differentiation     77
Resistogram typing     77
The slide agglutination technique     79
Serotyping of C. albicans by flow cytomerty     80
Phenotypic switching in C. albicans     81
Evaluation of phenotype switching in C. albicans     82
Extracellular enzymes secreted by C. albicans     83
Measurement of extracellular proteinase production by C. albicans     85
Measurement of extracellular proteinase produced by C. albicans (staib method)     86
Measurement of extracellular phospholipases of C. albicans     87
Germ-tube formation in C. albicans     88
Germ-tube formation assay     88
References     89
Analysis of Drug Resistance in Pathogenic Fungi   Gary P. Moran   Emmanuelle Pinjon   David C. Coleman   Derek J. Sullivan     93
Introduction     93
Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts      96
Method for the determination of minimum inhibitory concentrations (MICs) of antifungal agents for yeasts     97
Measurement of Rhodamine 6G uptake and glucose-induced efflux by ABC transporters     102
Measurement of rhodamine 6G uptake and glucose-induced efflux     102
Analysis of expression of multidrug transporters in pathogenic fungi     105
Analysis of point mutations in genes encoding cytochrome P-450 lanosterol demethylase     106
Qualitative detection of alterations in membrane sterol contents     108
Qualitative detection of alterations in membrane sterol contents     109
Overview     110
References     110
Animal Models for Evaluation of Antifungal Efficacy Against Filamentous Fungi   Eric Dannaoui     115
Introduction     115
Disseminated zygomycosis in non-immunosuppressed mice     118
Disseminated zygomycosis in non-immunosuppressed mice     118
Animal model of disseminated aspergillosis     125
Disseminated aspergillosis in neutropoenic mice     126
Study design for evaluation of antifungal combinations therapy in animal models     130
Study design for the evaluation of combination therapy in animal models     131
References     133
Proteomic Analysis of Pathogenic Fungi   Alan Murphy     137
Introduction     137
2D SDS-PAGE of protein samples     139
Protein digestion in preparation for mass spectrometry by MALDI-TOF     140
Peptide mass fingerprinting (PMF) by MALDI-TOF mass spectrometry     141
In-gel digestion     142
In-solution digestion     143
MALDI-TOF mass spectrometry     145
Preparation of matrix for MALDI-TOF     147
Peptide mass fingerprinting (PMF)     149
Post-source decay (PSD) and chemically assisted fragmentation (CAF)     150
Interpreting MALDI-TOF result spectra     152
Overview     156
References     157
Extraction and Detection of DNA and RNA from Yeast   Patrick Geraghty   Kevin Kavanagh     159
Introduction     159
The extraction of yeast DNA with the aid of phenol: chloroform     161
Whole-cell DNA extraction from C. albicans using phenol: chloroform     161
Rapid extraction of DNA from C. albicans colonies for PCR     164
Detection of yeast DNA using radio-labelled probes     165
DNA detection by Southern blotting     165
Extraction of whole-cell RNA using two different protocols      169
The extraction of whole-cell RNA from yeast using phenol: chloroform     170
Rapid extraction of whole-yeast-cell RNA     172
Detection and expression levels of specific genes by the examination of mRNA in yeast     174
Examining mRNA content as a means of investigating gene-expression profile by northern blot analysis     175
Examining mRNA content as a means of investigating gene-expression profile by RT-PCR analysis     176
References     179
Microarrays for Studying Pathogenicity in Candida Albicans   Andre Nantel   Tracey Rigby   Herve Hogues   Malcolm Whiteway     181
Introduction     181
DNA microarrays     182
Building a second-generation 2-colour long oligonucleotide microarray for C. albicans     183
Isolation of C. albicans RNA     185
Isolation of total RNA using the hot phenol method     186
Isolation of Poly-A+ mRNA     187
Determination of the efficiency of incorporation of the probe     192
Hybridization to DNA microarrays     194
Experiment design     196
Microarray-based studies in C. albicans     200
Conclusion     205
References     205
Molecular Techniques for Application with Aspergillus Fumigatus   Nir Osherov   Jacob Romano     211
Introduction     211
Preparation of knockout vectors for gene disruption and deletion in A. fumigatus     212
Preparation of knockout vectors for gene disruption and deletion in A. fumigatus     213
Transformation of A. fumigatus     217
Chemical transformation of A. fumigatus     218
Molecular verification of correct single integration (PCR-based)     220
Molecular verification of correct integration by PCR     221
General strategies for the phenotypic characterization of A. fumigatus mutant strains     223
General strategies for the phenotypic characterization of mutants     223
References     229
Promoter Analysis and Generation of Knock-out Mutants in Aspergillus Fumigatus   Matthias Brock   Alexander Gehrke   Venelina Sugareva   Axel A. Brakhage     231
Introduction     231
Site-directed mutagenesis of promoter elements     233
Site-directed mutation of promoter elements     233
lacZ as a reporter gene     236
lacZ as a reporter gene: discontinuous determination of [Beta]-galactosidase activity     237
lacZ as a reporter gene: continuous determination of [Beta]-galactosidase activity     239
Transformation of A. fumigatus     241
Transformation of A. fumigatus     242
Hygromycin B as a selection marker for transformation     246
Hygromycin B as a selection marker for transformation     247
pyrG as a selection marker for transformation     249
pyrG as a selection marker for transformation     249
URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions     251
URA-blaster (A. niger pyrG) as a reusable selection marker system for gene deletions/disruptions     253
References     255
Microarray Technology for Studying the Virulence of Aspergillus Fumigatus   Darius Armstrong-James   Thomas Rogers     257
Introduction     257
Isolation of RNA from A. fumigatus     259
Isolation of total RNA from A. fumigatus     260
Reverse transcription of RNA and fluorescent labelling of cDNA     263
Indirect labelling of cDNA with fluorescent dyes     264
Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing     266
Hybridization of fluorescent probes to DNA microarrays and post-hybridization washing     267
Image acquisition from hybridized microarrays      269
Image acquisition from hybridized microarrays     269
Microarray image analysis     270
Microarray image analysis     271
References     272
Techniques and Strategies for Studying Virulence Factors Cryptococcus Neoformans   Nancy Lee   Guilhem Janbon     275
Introduction     275
Construction of C. neoformans gene-disruption cassettes     278
Construction of C. neoformans gene-disruption cassettes     278
Genetic transformation of C. neoformans     283
Biolistic transformation of C. neoformans     283
Transformation via electroporation     286
Extraction of genomic DNA from C. neoformans     287
DNA for use in library construction and hybridization analysis     288
DNA for use in PCR     291
Screening and identification of deletion strains     292
Screening and identification of deletion strains     292
Measuring capsule size in C. neoformans     297
Measuring capsule size in C. neoformans     298
Purification of glucuronoxylomannan (GXM)     298
Purification of glucuronoxylomannan (GXM)     299
References     301
Genetic Manipulation of Zygomycetes   Ashraf S. Ibrahim   Christopher D. Skory     305
Introduction     305
Genetic tools to manipulate mucorales     306
Selectable markers used with mucorales fungi     307
Introduction of DNA used for transformation     308
Protoplasting of R. oryzae     309
Biolistic delivery system transformation of R. oryzae     313
A. tumefaciens-mediated transformation     316
Molecular analysis of transformants     319
Overview     322
References     323
Index     327