Preface
Contents
Contributors
Chapter 1: Quantifying the Mechanical Properties of Yeast Candida albicans Using Atomic Force Microscopy-based Force Spectrosc...
1 Introduction
2 Materials
2.1 Fungal Strain, Medium, and Solutions
2.2 Consumables
2.3 Microscope Specifications
3 Methods
3.1 Yeast Growth and Preparation
3.2 AFM Setup and Calibration
3.3 Force Spectroscopy Measurements
3.4 Data Preprocessing
3.5 Contact Mechanics Analysis
4 Notes
References
Chapter 2: Standardization of Galleria mellonella as an Infection Model for Malassezia furfur and Malassezia pachydermatis
1 Introduction
2 Materials
2.1 Larvae Preparation
2.2 Larval Inoculation and Survival and Melanization Evaluation
2.3 Fungal Burden Evaluation
2.4 Hemocytes Population Evaluation
2.5 Histological Procedure
3 Methods
3.1 Larvae Preparation
3.2 Larval Inoculation and Survival and Melanization Evaluation
3.3 Fungal Burden Evaluation
3.4 Hemocytes Population Evaluation
3.5 Histological Procedure
4 Notes
References
Chapter 3: Mouse Organotypic Brain Slice Cultures: A Novel Model for Studying Neuroimmune Responses to Cryptococcal Brain Infe...
1 Introduction
2 Materials
2.1 Cryptococcus neoformans Culture
2.2 Organotypic Culturing
2.2.1 Culture Trays
2.2.2 Decapitation and Brain Removal
2.3 Hippocampal Brain Slice Infection and Sample Collection
2.3.1 Stimulation
2.3.2 Sample Collection
2.4 Immunohistochemistry
3 Methods
3.1 Organotypic Culturing
3.1.1 Plate Preparation (Complete in a Biological Safety Cabinet)
3.1.2 Organotypic Culture Set-Up
3.1.3 Extracting the Brain
3.1.4 Hippocampal Removal
3.1.5 Slicing and Plating of the Hippocampal Slices
3.2 Culturing (Cryptococcus neoformans)
3.3 Infection of Organotypic Brain Slices with C. neoformans
3.3.1 Direct Stimulation of Brain Tissue with Whole-Cell C. neoformans
3.3.2 Sample Collection
3.4 Immunofluorescence Staining
3.4.1 Fixation, Blocking, and Primary Antibody Incubation (Day 1)
3.4.2 Secondary Antibody Incubation, Nuclear Staining, and Mounting (Day 2)
4 Notes
References
Chapter 4: Zebrafish Larvae as an Experimental Model of Cryptococcal Meningitis
1 Introduction
2 Materials
2.1 Preparing Streaking Plate and Pathogen Culture
2.2 Zebrafish Husbandry, Sorting, and Dechorinating
2.3 Zebrafish Injection with C. neoformans, Plating Inoculum and Mounting
3 Methods
3.1 Zebrafish Microinjections with C. neoformans
3.1.1 Making a Streaking Plate
3.1.2 Day 1: Zebrafish Husbandry and Marbling
3.1.3 Day 2: Obtaining Embryos and Sorting
3.1.4 Day 3: Dechorinating and Preparing Pathogen Culture
3.1.5 Day 4: Preparing Injection Inoculum
3.1.6 Day 4: Setting Up Needle and Anesthetizing Fish
3.1.7 Day 4: Injecting Zebrafish Larvae
3.1.8 Day 4: Plating Inoculum
3.2 Mounting for Widefield Fluorescence Microscopy
3.3 Screening and Mounting for Light Sheet Microscopy (see Note 13)
3.3.1 Screening
3.3.2 Mounting
3.4 Imaging
3.4.1 Widefield Set-Up Variations
3.4.2 Light Sheet Set-Up Variations (see Note 20)
3.5 Processing Data
4 Notes
References
Chapter 5: Immunological Analysis of Cryptococcal Meningoencephalitis in a Murine Model
1 Introduction
2 Materials
2.1 Cryptococcus neoformans Culture
2.2 Retro-Orbital Injections in Mice
2.3 Organ Collection
2.4 CFU Assay
2.5 Brain Leukocyte Isolation
2.6 Flow Cytometry Analysis Extracellular and Intracellular*
3 Methods
3.1 Cryptococcus neoformans Culture
3.2 Mice Infection (see Note 1)
3.2.1 Wash, Count, and Adjustment of C. neoformans Culture
3.2.2 Mouse Intravenous Injection (see Note 1)
3.2.3 Mice Monitoring Postinfection
3.3 Murine Coma and Behavioral Scale (MCBS) Adapted for Cryptococcal Meningoencephalitis
3.4 Collect Organs
3.4.1 Perfusion
3.4.2 Brain Collection Procedure
3.5 Colony Forming Unit (CFU) Plating Procedure
3.6 Brain Leukocyte Isolation
3.7 Flow Cytometry Analysis of Brain Immune Isolates
3.7.1 In vitro Stimulation of Brain Immune Cells for Intracellular Flow Cytometry (Optional)
3.7.2 Surface Staining of Brain Immune Cells for Flow Cytometry in 96-Well Plates
3.7.3 Staining for Intracellular Proteins
4 Notes
Bibliography
Chapter 6: Mouse Model of Latent Cryptococcal Infection and Reactivation
1 Introduction
2 Materials
2.1 LCNI Mouse Model Using ฮgcs1 Strain
2.2 LCNI Mouse Model Using UgCl223 or UgCl552 Strains
2.3 Cryptococcal Reactivation Mouse Model Using ฮgcs1 Strain
2.4 Cryptococcal Reactivation Mouse Model Using UgCl223 or UgCl552 Strains
2.5 Burden Analysis
2.6 Histological Analysis
3 Methods
3.1 Preparation of the Fungus
3.2 Infection Procedure
3.3 Monitoring and Confirmation of LCNI
3.3.1 Analysis of Fungal Burden
3.3.2 Histological Analysis
3.4 Reactivation of LCNI
3.4.1 Cryptococcal Reactivation Mouse Model Using ฮgcs1 Strain
3.4.2 Cryptococcal Reactivation Mouse Model Using UgCl223 or UgCl552 Strains
3.5 Expected Results
4 Notes
References
Chapter 7: Adoptive Transfer of Cryptococcus neoformans-Specific CD4 T-Cells to Study Anti-fungal Lymphocyte Responses In Vivo
1 Introduction
2 Materials
2.1 Animals
2.2 Tissue Preparation (see Note 3)
2.3 CD4 Isolation
2.4 Infection
3 Methods
3.1 Isolation of Lymph Nodes and Spleens from Donor Mice
3.2 Preparation of Single Cell Suspension from Donor Lymphoid Organs
3.3 CD4 T-cell Isolation
3.4 Proliferation Dye Staining
3.5 Check of the Purity of CD4 T-Cells and the Staining of Proliferation Dye
3.6 Transfer and Infection
3.7 Tissue Preparation: Analysis
3.7.1 Digestion and Preparation of Brain
3.7.2 Digestion and Preparation of Lung
3.8 Flow Cytometry Analysis
4 Notes
References
Chapter 8: Meningeal Whole Mounts for Imaging CNS Fungal Infection
1 Introduction
2 Materials
2.1 Dissection
2.2 Immunostaining
3 Methods
3.1 Tissue Collection
3.2 Dissecting the Meninges
3.3 Immunostaining
3.4 Mounting
4 Notes
References
Chapter 9: An Antibiotic-Free Model of Candida albicans Colonization of the Murine Gastrointestinal Tract
1 Introduction
2 Materials
2.1 Preparation of Fructose Water
2.2 Growth of Candida albicans
2.3 Stool Collection and Processing
3 Methods
3.1 Candida albicans Culture
3.2 Candida albicans Colonization
3.3 Monitoring of Candida albicans Colonization Levels
4 Notes
References
Chapter 10: An Experimental Model of Chromoblastomycosis Caused by Fonsecaea sp. Species
1 Introduction
2 Materials
2.1 Preparation of Fungal Solution
2.2 Suspension of Infection
2.3 Animals and Experimental Design
2.4 Morphometry
2.5 Fungal Burden
2.6 Histopathology
2.7 Lymphocytes Isolation from Draining Lymph Node (dLN) and Footpad
3 Methods
3.1 Preparation of Fungal Solution
3.1.1 Fungal Propagules
3.1.2 Muriform Cells
3.2 Suspension of Infection
3.3 Animals and Experimental Design
3.3.1 Rat Infection Model
3.3.2 Mouse Infection Model
3.4 Analysis Checkpoints
3.5 Morphometry
3.6 Fungal Burden
3.7 Histopathology
3.8 Lymphocytes Isolation from Draining Lymph Node (dLN) and Footpad
3.8.1 Lymph Node
3.8.2 Footpad Tissue
4 Notes
References
Chapter 11: Modeling Chronic Coccidioidomycosis in Mice
1 Introduction
2 Materials
2.1 Intranasal Infection
2.2 Terminal Exsanguination
2.3 Organ Collection
2.4 Preparation of Lungs for Histopathology
3 Methods
3.1 Preparation of Infecting Suspension
3.2 Intranasal Infection
3.3 Postinfection Verification of Infectious Dose
3.4 Monitoring Mice
3.5 Collection of Tissue for Quantitative Fungal Culture or Flow Cytometry
3.6 Terminal Exsanguination (see Note 26)
3.7 Collection of Lungs for Histopathology
4 Notes
References
Chapter 12: Mouse Models of Phaeohyphomycosis
1 Introduction
2 Materials
2.1 Subcutaneous Phaeohyphomycosis Mouse Model
2.2 Footpad Swelling Rate Assay
2.3 Fungal Burdens Measurement
2.4 Histopathological Analysis
2.5 Local Immune Responses Study
3 Methods
3.1 Conidia Suspension Preparation
3.2 Establishment of Subcutaneous Phaeohyphomycosis Mouse Model
3.3 Footpad Swelling Rate Assay
3.4 Fungal Burdens Measurement in the Infected Footpads
3.5 Survival Rates Assay
3.5.1 Expected Results
3.6 Histopathological Analysis
3.7 Local Immune Responses Study
3.7.1 qRT-PCR Analysis for Relative Cytokine and Chemokine Gene Expression in Footpad Skin Tissue
3.7.2 Measurement for Proteins of Cytokine and Chemokine in Footpad Homogenates
3.7.3 Local Neutrophils Infiltration by FACS Analysis
4 Notes
References
Chapter 13: Genetic Mouse Models of Pneumocystis Pneumonia
1 Introduction
2 Materials
2.1 Pneumocystis Murina Propagation In Vivo
2.2 Pneumocystis Murina Infections
2.3 Tissue Collection and Analysis
3 Methods
3.1 Pneumocystis Murina Propagation In Vivo
3.2 Route of Experimental Infection
3.2.1 Natural Transmission/Co-Housing Model
3.2.2 Intranasal Instillation
3.2.3 Oropharyngeal Instillation
3.2.4 Transtracheal Injection
3.3 IRIS Mouse Model
3.4 Experimental Parameters
3.4.1 BAL Fluid Collection for P. Murina Detection
3.4.2 Lung Histopathology
3.4.3 RNA Isolation and Quantitative PCR for P. Murina
3.4.4 Plethysmography (for IRIS)
4 Notes
References
Chapter 14: Mouse Models of Mucormycosis
1 Introduction
2 Materials
2.1 Immunosuppression by Corticosteroids
2.2 Induction of Leukopenia Using Cyclophosphamide
2.3 Induction of Diabetic Ketoacidosis (DKA)
2.4 Intranasal Infection
2.5 Clinical Monitoring and Humane Endpoints
3 Methods
3.1 Experimental Planning and Acclimatization of Animals
3.2 Immunosuppression by Corticosteroids
3.3 Induction of Leukopenia Using Cyclophosphamide
3.4 Induction of Diabetic Ketoacidosis (DKA) Using Streptozotocin
3.5 Intranasal Infection
3.6 Clinical Monitoring and Humane Endpoints
4 Notes
References
Chapter 15: Bioluminescence Imaging, a Powerful Tool to Assess Fungal Burden in Live Mouse Models of Infection
1 Introduction
2 Materials
2.1 Fungal Strains
2.2 Fungal Culture and Harvesting
2.3 Animals and Housing
2.4 Immunosuppression to Allow for invasive A. fumigatus Infection
2.5 Orotracheal Inoculation
2.6 Anesthesia
2.7 BLI
2.8 Tissue Isolation at Endpoint
3 Methods
3.1 Immunosuppression for Neutropenic Model of IPA
3.2 Fungal Culture and Spore Harvesting
3.3 Inducing Mouse Model of Infection by Orotracheal Inoculation
3.4 BLI Acquisition and Quantification
3.5 Experiment Termination
4 Notes
References
Chapter 16: Microcomputed Tomography to Visualize and Quantify Fungal Infection Burden and Inflammation in the Mouse Lung Over...
1 Introduction
2 Materials
2.1 Animal Models
2.1.1 Mouse model of IPA
2.1.2 Mouse model of Pulmonary cryptococcosis
2.2 Orotracheal Inoculation
2.3 Anesthesia
2.4 Microcomputed Tomography (ฮผCT)
2.5 Software
3 Methods
3.1 Experimental Fungal Infection (Orotracheal Instillation)
3.2 ฮผCT Acquisition for Skyscan 1278
3.3 ฮผCT Scan Reconstruction
3.4 ฮผCT Data Visualization
3.5 ฮผCT Data Quantification
3.5.1 Semi-quantitative Scoring of ฮผCT Scans Based on Visual Observation
3.5.2 Quantification of ฮผCT-Derived Biomarkers
4 Notes
References
Index