دانلود کتاب Trypanosomatids: Methods and Protocols

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Preface
Contents
Contributors
Part I: Isolation, Culturing, and Genetic Manipulation of Trypanosomatids
Chapter 1: Field Isolation and Cultivation of Trypanosomatids from Insects
1 Introduction
2 Materials
3 Methods
3.1 Catching Techniques
3.2 Keeping the Hosts
3.3 Dissection and Slide Preparation
3.4 Light Microscopy
3.5 Division of the Sample
3.6 Preparation of Slides
3.7 Preparation of Vials and Media for Primo-Cultures
3.8 Axenization
3.9 Culture Cloning
3.10 Cryopreservation
3.11 DNA Analysis
4 Notes
References
Chapter 2: Culturing and Transfection of Pleomorphic Trypanosoma brucei
1 Introduction
2 Materials
2.1 Trypanosoma brucei brucei Strains
2.1.1 AnTat 1.1E
2.1.2 AnTat 1.1E ``Paris´´
2.1.3 AnTat 1.1 ``Munich´´
2.1.4 AnTat 1.1 90-13
2.2 T. brucei Culture Media and Transfection Solutions
2.2.1 Modified HMI-9
2.2.2 HMI-9 with Methylcellulose
2.2.3 HMI-9 with Agarose
2.2.4 Electroporation Buffer
2.3 Equipment for Cultivation of T. brucei
2.4 Materials and Equipment for Electroporation of T. brucei
3 Methods
3.1 Cultivation of Pleomorphic Trypanosoma brucei brucei Strains
3.1.1 Cultivation of T. brucei on Agarose Plates
Preparation of Agarose Plates (Adapted from, Modified by and)
Spreading of T. brucei Bloodstream Forms on Agarose Surface
3.1.2 Cultivation of T. brucei in Methylcellulose HMI-9 Medium
Preparation of Methylcellulose HMI-9 (Adapted from)
3.1.3 Cultivation of Matrix-Independent T. brucei Strains
3.2 Transfection of Pleomorphic Trypanosoma brucei brucei Strains
3.2.1 Preparation of DNA for Transfection
3.2.2 Transfection of Matrix-Dependent Strains
From In Vitro Culture in Methylcellulose HMI-9 (Adapted from)
Transfection of T. brucei Derived from Animal Blood
3.2.3 Transfection of Matrix-Independent Strains
3.3 Recommendation for Strain Selection
4 Notes
References
Chapter 3: In Vitro Culture for Differentiation Simulation of Leishmania spp.
1 Introduction
2 Materials
2.1 Promastigote Growth Medium
2.2 Amastigote Growth Medium
2.3 Serum for Amastigote Media
3 Methods
3.1 Protocol for Axenic Promastigote-to-Amastigote Differentiation
3.2 Morphological Assessment of Differentiation
3.3 Cell Aggregation Is Essential for Differentiation Progression
3.4 Quality Control
4 Notes
References
Chapter 4: Tsetse Fly Transmission Studies of African Trypanosomes
1 Introduction
2 Materials
2.1 Tsetse Infection
2.2 Tsetse Dissection
2.3 Isolation of Trypanosomes
2.4 Tsetse Salivary Exudate Sampling
3 Methods
3.1 Infecting Tsetse with Trypanosomes
3.2 Dissection of Infected Tsetse
3.2.1 Examining the Gut and Salivary Glands
3.2.2 Examining the Proboscis and Cibarium
3.3 Isolation of Trypanosomes
3.3.1 Isolating Trypanosomes from the Midgut
3.3.2 Isolating Trypanosomes from the Salivary Glands
3.3.3 Isolating Trypanosomes from the Proventriculus
3.3.4 Isolating Trypanosomes from the Foregut Via Salivary Exudate
4 Notes
References
Chapter 5: Infecting Triatomines with Trypanosomes
1 Introduction
2 Materials
2.1 Culture Media and Solutions
2.1.1 Liver Infusion Tryptose (LIT) Medium
2.1.2 Novy, McNeal, and Nicolle (NNN) Medium
2.1.3 Hemolymph Anticoagulant Solution
2.2 Insect Feeding
2.3 Insect Inoculum
3 Methods
3.1 Parasite Culture Techniques
3.2 Infection of R. prolixus with Trypanosomes
3.2.1 Infection with Cultured Epimastigotes
3.2.2 Infection with Blood Trypomastigotes
Trypanosoma cruzi
Trypanosoma rangeli
3.3 Confirming the Infection
3.3.1 Trypanosoma cruzi
3.3.2 Trypanosoma rangeli
3.4 Maintaining Trypanosome Infectivity
4 Notes
References
Part II: Trypanosomatid Genome-Wide Analyses
Chapter 6: High-Throughput Sequencing for Trypanosome Transcriptome Characterization
1 Introduction
1.1 General Considerations
1.2 Transcriptomes of Cultured Trypanosomes
1.3 Preparing RNA from Trypanosomes in Animals
1.4 Trypanosomes from Clinical Samples
1.5 Selecting RNA for Sequencing
1.6 Sequence Analysis: Aligning and Counting Reads
1.7 Differential Gene Expression Analysis: Comparing Conditions
1.8 Visualizing Relationships Between Datasets and Finding Coregulated Genes
2 Materials
2.1 Trypanosomes from Culture
2.2 Trypanosomes from Blood and Clinical Samples
2.2.1 Growing Parasites in Rodents
2.2.2 Purification Using DEAE Cellulose
2.2.3 Trypanosomes from Clinical Samples
2.2.4 Trypanosomes from CSF
2.3 Removal of rRNA by RNase H Digestion
2.4 Sequence Data Analysis
3 Methods
3.1 Trypanosomes from Culture
3.2 Trypanosomes from Blood and Clinical Samples
3.2.1 Growing Parasites in Rats
3.2.2 Purification of Trypanosomes from Blood by DEAE-Cellulose Chromatography
3.2.3 Infected Human Blood
3.2.4 Human CSF
3.3 rRNA Depletion Using Oligonucleotides and RNase H
3.4 Sequence Analysis
3.4.1 Sequence Alignment
3.4.2 Differential Expression Analysis
3.4.3 Visualizing the Differences Between Samples Using ClusterViewer
3.4.4 Calculating the Average Number of mRNAs per Cell
4 Notes
References
Chapter 7: Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei
1 Introduction
2 Materials
2.1 Polysome Gradients
2.2 Metabolic Labeling to Measure Protein Synthesis
3 Methods
3.1 Polysome Analysis Using Sucrose Gradients
3.1.1 Preparation of Sucrose Gradients
3.1.2 Lysate Preparation and Gradient Loading
3.1.3 Fractionation (See Note 7)
3.1.4 RNA Analysis
3.1.5 Proteins from the Fractions
3.2 Pulse Labeling
4 Notes
References
Chapter 8: Immunoprecipitation for the Analysis of Macromolecular Complexes in Trypanosoma cruzi
1 Introduction
2 Materials
2.1 Materials
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Cell Preparation
3.2 Cell Lysis
3.3 Bead Preparation
3.4 Immunoprecipitation
4 Notes
References
Chapter 9: Analysis of the In Vivo Translation Process in Trypanosoma cruzi Using Ribosome Profiling
1 Introduction
2 Materials
2.1 Materials
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Cell Culture
3.2 Cell Lysis
3.3 Polysome Isolation
3.4 Digestion
3.5 Ribosome Footprinting Isolation
3.6 RNA Size Selection
4 Notes
References
Chapter 10: Proteome-Wide Quantitative Phosphoproteomic Analysis of Trypanosoma brucei Insect and Mammalian Life Cycle Stages
1 Introduction
2 Materials
2.1 SILAC Labeling of Cultures and Cell Lysis
2.2 Protein Solubilization and Tryptic Digest
2.3 Desalting Using a C18 Column
2.4 Fe-Immobilized Metal Ion Affinity Chromatography (Fe-IMAC)
2.5 High pH Fractionation
2.6 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
2.7 Data Analysis
3 Methods
3.1 SILAC Labeling of Cultures and Cell Lysis
3.2 Protein Solubilization and Digestion
3.3 Desalting Using a C18 Cartridge
3.4 Fe-IMAC to Separate Peptides from Phosphopeptides
3.5 High pH Fractionation
3.6 Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)
3.7 Data Analysis
4 Notes
References
Chapter 11: Genome-Wide Proteomics and Phosphoproteomics Analysis of Trypanosoma cruzi During Differentiation
1 Introduction
2 Materials
3 Methods
3.1 T. cruzi Cultivation
3.2 T. cruzi Metacyclogenesis and Purification of the Metacyclic Form
3.3 Sample Preparation for Proteomics and Phosphoproteomics
3.3.1 Filter-Aided Sample Preparation (FASP)
3.3.2 High pH Reversed-Phase Fractionation
3.3.3 Phosphopeptide Enrichment
3.4 Proteomics and Phosphoproteomics Analysis by LC-MS/MS
3.5 Proteomics and Phosphoproteomics Data Analysis
3.5.1 Processing MS Data with MaxQuant
3.5.2 Analyzing MS Data Using Perseus
Total Proteome Analysis
Phosphoproteome Analysis
4 Notes
References
Chapter 12: Genome-Wide Proteomics and Phosphoproteomics Analysis of Leishmania spp. During Differentiation
1 Introduction
2 Materials
2.1 Promastigote Growth Medium
2.2 Amastigote Growth Medium
2.3 Serum for Amastigote Medium
2.4 Cell Lysis and Protein Extraction
2.5 SDS-PAGE
2.6 In-Solution Protein Digestion
2.7 Digested Peptide Cleanup Using C18 Sep-Pak Columns
2.8 TMT Labeling of Peptides
2.9 Labeled Peptide Sample Fractionation
2.10 Phosphopeptide Enrichment
2.10.1 Titanium Dioxide (TiO2) Enrichment
2.10.2 Immobilized Metal Affinity Chromatography (IMAC) Enrichment
2.11 Desalting Using STAGE Tips
2.12 LC-MS/MS Analysis
2.13 Mass Spectrometry Data Analysis
3 Methods
3.1 Principle of TMT Labeling Assay
3.2 Protocol for Axenic Promastigote-to-Amastigote Differentiation
3.3 Cell Lysis and Protein Extraction
3.4 In-Solution Protein Digestion
3.5 Digested Peptide Cleanup Using C18 Sep-Pak Columns
3.6 TMT Labeling of Peptides
3.7 Labeled Peptide Sample Fractionation Using Basic pH Reverse Phase Liquid Chromatography (bRPLC)
3.8 Phosphopeptide Enrichment
3.8.1 Titanium Dioxide (TiO2) Enrichment
3.8.2 Immobilized Metal Affinity Chromatography (IMAC) Enrichment
3.9 C18 Stage-Tips Cleanup
3.10 LC-MS/MS Analysis
3.11 Mass Spectrometry Data Analysis
4 Notes
References
Chapter 13: CRISPR/Cas9 Technology Applied to the Study of Proteins Involved in Calcium Signaling in Trypanosoma cruzi
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Amplification and Cloning of sgRNA
2.3 Amplification and Purification of Donor DNA
2.4 Cell Transfections
2.5 Verification of Genome Editing
2.6 Gene KO Complementation
2.7 Mitochondrial Calcium Uptake
3 Methods
3.1 Cell Culture
3.2 Endogenous Gene Tagging
3.2.1 Selection of Protospacers
3.2.2 Amplification and Cloning of sgRNA
3.2.3 Amplification and Purification of Donor DNA
3.2.4 Cell Transfections
3.2.5 Verification of Endogenous Gene Tagging
3.3 Gene Knockout
3.3.1 Selection of Protospacers for Gene KO
3.3.2 Amplification and Cloning of sgRNA for Gene KO
3.3.3 Amplification and Purification of Donor DNA for Gene KO
3.3.4 Cell Transfections
3.3.5 Verification of Gene KO
3.4 Gene Complementation of Knockout Mutants Obtained by CRISPR/Cas9
3.5 Mitochondrial Calcium Uptake for Phenotype Analysis of CRISPR/Cas9 Mutant Cell Lines Involved in Calcium Homeostasis
4 Notes
References
Chapter 14: Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania
1 Introduction
2 Materials
2.1 Molecular Cloning
2.2 Leishmania Strains and Culture Medium
2.3 Parasite Transfection
3 Methods
3.1 Selection of CRISPR Targeting Site(s)
3.2 gRNA Design
3.3 Clone a gRNA Coding Sequence into the Leishmania CRISPR Vector pLdCN
3.3.1 The Traditional Two-Step Digestion and Ligation Cloning Protocol
3.3.2 The Single-Step Digestion-Ligation Cloning Protocol
3.4 Clone Two gRNA Guide Coding Sequences into Leishmania CRISPR Vector pLdCN
3.4.1 The Cloning Protocol Using PCR Generated DNA Cassette
3.4.2 The Single-Step Digestion-Ligation Cloning Protocol for Two gRNA
3.5 Transfection of Leishmania with pLdCN Plasmid or Donor DNA
3.6 Isolate the CRISPR Edited Mutants
3.7 Generate Gene Deletion Mutants Without Using a Donor
3.8 Generate Gene Edited Mutants by Using an Oligonucleotide Donor
3.9 Generate Gene Disrupted Mutants by Using an Antibiotic Resistance Selection Marker Donor
3.10 Delete Multicopy Family Genes
3.11 Target Essential Leishmania Genes
3.12 Gene Tagging
3.13 Gene Complementation
3.14 Generate Targeted Chromosome Translocations
4 Notes
References
Chapter 15: Next-Generation Analysis of Trypanosomatid Genome Stability and Instability
1 Introduction
2 Materials
2.1 MFA-seq Analysis of Replication Dynamics
2.1.1 Cell Preparation and Staining with Propidium Iodide
2.1.2 Cell Sorting, DNA Extraction, and NGS
2.1.3 Genomic DNA Extraction and Sequencing
2.1.4 Marker Frequency Analysis
2.2 ChIP-seq Analysis of γH2A Distribution
2.2.1 Chromatin Immunoprecipitation
2.2.2 Library Preparation
2.2.3 qPCR
2.2.4 Data Analysis
2.3 Next Generation Analysis of Instability
2.3.1 Hardware
2.3.2 Datasets
2.3.3 Software (See Note 29)
3 Methods
3.1 Mapping Replication Initiation by MFA-seq
3.1.1 Cell Preparation and Staining with Propidium Iodide-T. brucei Procyclic Cell Forms and L. major/L. mexicana Promastigote...
3.1.2 Cell Preparation and Staining with Propidium Iodide-T. brucei Bloodstream Form Cells
3.1.3 Cell Sorting into G1, S, and G2/M Phase Subpopulations
3.1.4 gDNA Extraction from the G1, S and G2/M Phase Subpopulations
3.1.5 Sequencing Library Preparation
3.1.6 Marker Frequency Analysis
3.2 Localization of DNA Damage by γH2A ChIP-seq
3.2.1 Chromatin Extraction and Enzymatic Shearing
3.2.2 DNA Clean Up, Quantification and Shearing Check
3.2.3 Chromatin-Immunoprecipitation (ChIP)
3.2.4 ChIP-qPCR
3.2.5 ChIP-seq
3.3 Next Generation Sequencing to Examine Genome Variation
3.3.1 Download Data Sets
3.3.2 Run FastQC to Evaluate the Read Library Quality
3.3.3 Run Trimmomatic
3.3.4 Mapping the Reads to the Reference Genome
3.3.5 Filter Alignment File by Mapping Quality and Convert .sam File to .bam
3.3.6 Sort .bam File
3.3.7 Estimate the Mean Genome Coverage
3.3.8 Estimate the Chromosomal Somy by RDC
3.3.9 Generate a CCNV Plot
3.3.10 Restrict the Somy Estimation to Coding Sequences (CDSs) Regions
3.3.11 Chromosomal Somy Estimations Based on Allele Frequency Analysis
4 Notes
References
Part III: Molecular Biology and Genetics
Chapter 16: Transcription Factor Analysis in Trypanosomatids
1 Introduction
2 Materials
2.1 Extract Preparation
2.2 In Vitro Transcription Analysis
2.2.1 In Vitro Transcription Reaction and RNA Preparation
2.2.2 Factor Depletion from Extract
2.2.3 Antibody-Mediated Transcription Inhibition
2.2.4 Radiolabeling of Oligonucleotides and DNA Marker
2.2.5 Primer Extension Reaction and Denaturing Polyacrylamide Gel Electrophoresis (PAGE)
2.3 Promoter Pull Down Assay
3 Methods
3.1 Extract Preparation
3.1.1 Small-Scale WCE Preparation
3.1.2 Large-Scale WCE Preparation
3.1.3 Nuclear Extract Preparation (Large Scale)
3.2 In Vitro Transcription Analysis
3.2.1 In Vitro Transcription Reaction and RNA Preparation
3.2.2 Factor Depletion from Extract
3.2.3 Antibody-Mediated Transcription Inhibition
3.2.4 Radiolabeling of Oligonucleotides and DNA Marker
3.2.5 Primer Extension Reaction and Denaturing PAGE
3.3 Promoter Pull-Down Assay
4 Notes
References
Chapter 17: Identifying Trypanosome Protein-RNA Interactions Using RIP-Seq
1 Introduction
2 Materials
2.1 Cell Line
2.2 Cell Harvesting and In Vivo UV Cross-Linking
2.3 RNA Immunoprecipitation
3 Methods
3.1 Sample Collection and In Vivo UV Cross-Linking
3.2 Cell Lysis and RNA Immunoprecipitation
3.3 Important Controls
3.4 Computational Analysis
3.5 Motif Discovery
4 Notes
References
Chapter 18: The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Construction of a CAT Reporter-Expressing Cell Line
3.2 Important Controls
3.3 Assay of Chloramphenicol Acetyltransferase (CAT Assay)
3.3.1 Radioactive Kinetic Assay
3.3.2 CAT Activity Quantification
4 Notes
References
Chapter 19: RNA-Binding Proteins and Their Targets in Trypanosoma brucei: Single Nucleotide Resolution Using iCLIP and iCLAP
1 Introduction
2 Materials
2.1 iCLIP Buffers
2.2 iCLAP Buffers
2.3 UV-Cross Linking
2.4 SDS-PAGE and Nitrocellulose Transfer
2.5 RNA Isolation
2.6 Reverse Transcription
2.7 cDNA Isolation and PCR Amplification
2.8 Trypanosome Culture
3 Methods
3.1 UV Cross-Linking of T. brucei (Fig. 1): Day 0
3.2 iCLIP (In Vivo Cross-Linking and Immunoprecipitation): Day 1 (Fig. 2)
3.2.1 Lysate Preparation
3.2.2 Partial RNA Digestion
3.2.3 Bead Preparation
3.2.4 Immunoprecipitation
3.3 iCLAP Protocol (In Vivo Cross-Linking and Affinity Purification Protocol)
3.3.1 Preparation of the Lysate: Day 1 (Fig. 2)
3.3.2 Partial RNA Digestion
3.3.3 Preparation of IgG Sepharose Beads
3.3.4 IgG Sepharose-Based Affinity Purification
3.3.5 Preparation of His-Dynabeads
3.3.6 His Dynabeads-Based Affinity Purification
3.4 SDS-PAGE and Transfer of Protein-RNA Complexes to Nitrocellulose Membrane: Day 2
3.5 RNA Isolation: Day 3
3.6 Reverse Transcriptase: Day 4
3.7 Size Selection of cDNA: Day 5
3.8 cDNA Circulation and Linearization: Day 6
3.9 PCR Amplification: Day 7
4 Notes
References
Chapter 20: In Vivo Tethering System to Isolate RNA-Binding Proteins Regulating mRNA Decay in Leishmania
1 Introduction
2 Materials
2.1 Generating Leishmania Stable Transfectants Co-expressing the MS2 Coat Protein and the Reporter LUC-2MS2-SIDER2 3′UTR or th...
2.2 Confirmation of the MS2 Coat Protein Expression by Western Blotting
2.3 Coimmunoprecipitation of the MS2 Coat Protein Complex Upon Tethering to the Reporter LUC-2MS2-SIDER2 3′UTR mRNA
3 Methods
3.1 Generating Leishmania Stable Transfectants Co-expressing the MS2 Coat Protein and the Reporter LUC-2MS2-SIDER2 3′UTR or th...
3.2 Confirmation of the MS2 Coat Protein Expression by Western Blotting
3.3 Co-immunoprecipitation of the MS2 Coat Protein Complex Upon Tethering to the Reporter LUC-2MS2-SIDER2 3′UTR mRNA
4 Notes
References
Chapter 21: Forward Genetics in African Trypanosomes
1 Introduction
2 Materials
2.1 T. brucei Culturing
2.2 T. brucei Library Strain Setup
2.3 Library Sequencing and Mapping
3 Methods
3.1 Preparation of Plasmid DNA for RNAi Library Strain Setup
3.2 T. brucei RNAi Library Strain Setup
3.3 Running an RNAi Library Screen-Selection by Drug Resistance
3.4 Running a RNAi Library Screen-Selection by Toxin
3.5 Running an RNAi Library Screen-Selection by Reporter Construct
3.6 High-Throughput Sequencing-RIT-Seq
3.7 Analyzing Sequence Data
4 Notes
References
Chapter 22: Analysis of Base Excision and Single-Strand Break Repair Activities in Trypanosomatid Extracts
1 Introduction
2 Materials
2.1 Preparation of Whole Cell Extracts
2.2 Preparation of Oligonucleotide Substrates
2.3 DNA Repair Activity Assays
2.4 Denaturing Polyacrylamide Gel Electrophoresis
3 Methods
3.1 Preparation of Cell Extracts
3.2 Preparation of Oligonucleotide Substrates
3.3 In Vitro DNA Repair Assays
3.4 Denaturing Polyacrylamide Gel Electrophoresis
4 Notes
References
Part IV: Cell Biology
Chapter 23: Light Microscopy in Trypanosomes: Use of Fluorescent Proteins and Tags
1 Introduction
2 Materials
3 Methods
3.1 Procyclic Form Cell Preparation
3.1.1 Preparation of Live Procyclic Form Trypanosomes for Imaging
3.1.2 Preparation of Methanol Fixed Procyclic Form Trypanosomes for Imaging
3.1.3 Preparation of Formaldehyde Fixed Procyclic Form Trypanosomes for Imaging
3.1.4 Preparation of Methanol Fixed Procyclic Form Cytoskeleton for Imaging
3.2 Bloodstream Form Cell Preparation
3.2.1 Preparation of Bloodstream Trypanosomes for Live Cell Imaging
3.2.2 Methanol Fixed Bloodstream Form
3.2.3 Preparation of Formaldehyde Fixed Bloodstream Form Trypanosomes for Imaging
3.3 Immunofluorescence of Fixed Trypanosomes
3.4 Microscopy of Trypanosomes (See Notes 22-25)
4 Notes
References
Chapter 24: ImageJ for Partially and Fully Automated Analysis of Trypanosome Micrographs
1 Introduction
2 Materials
2.1 Software
2.2 Images
3 Methods
3.1 Loading and Running ImageJ Macros
3.1.1 ImageJ Macro Syntax
3.1.2 Image Handling
3.2 Semiautomated Image Preparation for Presentation or Publication
3.2.1 Semiautomated Cell Rotation and Cropping
3.2.2 Automated Contrast
3.2.3 Automated Saving of Raw and Composite Images
3.3 Automated Cell Cycle Analysis
3.3.1 Thresholding Trypanosomes from Phase Contrast
3.3.2 Quantitation of Fluorescence Intensity
3.3.3 Kinetoplast and Nucleus Counts
3.4 Automated Quantitative Analyses
3.4.1 Quantitative Colocalization
3.4.2 Point Spread Function Fitting for Superresolved Analysis
4 Notes
References
Chapter 25: Motility Analysis of Trypanosomatids
1 Introduction
2 Materials
3 Methods
3.1 Motility Analysis
3.2 Time-Dependent Tomography
4 Notes
References
Chapter 26: Electron Microscopy Techniques Applied to Symbiont-Harboring Trypanosomatids: The Association of the Bacterium wit...
1 Introduction
2 Materials
2.1 Freeze Fracture and Deep Etching
2.1.1 Equipment
2.1.2 Sample Processing
2.2 High-Pressure Freezing
2.2.1 Equipment
2.2.2 Sample Processing
2.3 Freeze-Substitution
2.3.1 Equipment
2.3.2 Sample Processing
2.4 Focused Ion Beam
2.4.1 Equipment and Software
2.4.2 Sample Processing
2.5 Electron Tomography
2.5.1 Equipment and Software
2.5.2 Sample Processing
3 Methods
3.1 Freeze-Fracture (Fig. 9)
3.2 Quick Freeze and Deep Etching
3.3 High-Pressure Freezing
3.4 Freeze Substitution
3.4.1 Using a Commercial Freeze Substitution System
3.4.2 Using a Homemade System (see Note 23)
3.5 Focused Ion Beam (Fig. 5)
3.6 Electron Tomography (Fig. 6)
4 Notes
References
Chapter 27: Airyscan Superresolution Microscopy to Study Trypanosomatid Cell Biology
1 Introduction
2 Materials
2.1 Preparation of Poly-l-Lysine-Coated Glass Slides
2.2 Preparation of Cells for Live-Cell Microscopy
2.3 Preparation of Fixed Cells for Microscopy
2.4 Preparation of Cell Cytoskeletons for Microscopy
2.5 Preparation of Isolated Flagella for Microscopy
2.6 Staining Cells with MitoTracker Deep Red
2.7 Immunofluorescence
2.8 Mounting of Fixed Cell Preparation Prior to Microscopy
3 Methods
3.1 Preparation of Poly-l-Lysine-Coated Glass Slides
3.2 Preparation of Cells for Live-Cell Microscopy
3.3 Preparation of Fixed Cells for Microscopy
3.4 Preparation of Cell Cytoskeletons for Microscopy
3.5 Preparation of Isolated Flagella for Microscopy
3.6 Staining Cells with MitoTracker Deep Red
3.7 Immunofluorescence
3.8 Mounting of Fixed Cell Preparation Prior to Microscopy
3.9 Configuring the Zeiss LSM880 with Airyscan for Superresolution Imaging
4 Notes
References
Chapter 28: All You Ever Wanted to Know About APOL1 and TLFs and Did Not Dare Ask
1 Introduction
2 Materials
2.1 Trypanosome Lytic Factor 1 and 2 Purification
2.1.1 Chemicals and solutions
2.1.2 Equipment
2.1.3 Antibodies
2.2 Recombinant Apolipoprotein L1 Protein Purification
2.2.1 Buffer Solutions
2.2.2 Equipment
3 Methods
3.1 Separation of TLF1 and TLF2
3.2 TLF Affinity Purification Column Preparation
3.3 TLF1 Purification
3.4 TLF2 Purification
3.5 Final TLF Quality Checks
3.6 Transformation of Bacteria with APOL1 Plasmid
3.7 Bacterial Growth and Protein Expression
3.8 Isolation of Inclusion Bodies
3.9 Extraction of rAPOL1 from Inclusion Bodies
3.10 Fast Protein Liquid Chromatography
4 Notes
References
Chapter 29: Isolation of Leishmania Promastigote Flagella
1 Introduction
2 Materials
2.1 Deflagellation and Density Gradient Centrifugation
3 Methods
3.1 Preparing Cells
3.2 Deflagellation
3.3 Separation of Flagella and Deflagellated Cell Bodies
4 Notes
References
Chapter 30: Gel-Based Methods for the Investigation of Signal Transduction Pathways in Trypanosoma brucei
1 Introduction
2 Materials
2.1 PCR/Mutagenesis
2.2 Bacterial Transformation/Culture
2.3 Insect Cell Culture/Infection/Pulldown
2.4 Kinase Assay
2.5 Phos-Tag Assay
2.5.1 Sample Preparation
2.6 SDS-PAGE and Western Blot
2.6.1 General
2.6.2 Kinase Assays
2.6.3 Phos-Tag Aassay
3 Methods
3.1 Site-Directed Mutagenesis
3.1.1 PCR and DpnI Digestion
3.1.2 Bacterial Transformation
3.2 SF9 Insect Cells Expression System
3.2.1 Transformation
3.2.2 Transfecting Insect Cells
3.2.3 Amplifying Baculoviral Stock
3.2.4 Expression and Purification of the Recombinant Protein
3.3 Kinase Assays
3.3.1 ``Cold´´ Kinase Assay
3.3.2 ``Hot´´ Kinase Assay
3.3.3 SDS-PAGE and Transfer Onto PVDF Membrane
3.3.4 Phosphotransferase Activity Measurement
3.4 Phos-Tag Assay of Phosphorylated Proteins
3.4.1 Sample Prep
3.4.2 SDS PAGE Using Mini Protean Gel Equipment (Bio-Rad)
4 Notes
References
Chapter 31: Methods to Investigate Signal Transduction Pathways in Trypanosoma cruzi: Cyclic Nucleotide Phosphodiesterases Ass...
1 Introduction
2 Materials
2.1 Parasite Culture and Extracts
2.2 Phosphodiesterase Assay
3 Methods
3.1 Trypanosoma cruzi Culture and Extracts Preparation
3.2 Phosphodiesterase Assay
4 Notes
References
Chapter 32: Methods for the Investigation of Trypanosoma cruzi Amastigote Proliferation in Mammalian Host Cells
1 Introduction
1.1 Multiplexed Plate Assay to Measure Intracellular Parasite and Host Cell Abundance
1.2 Fluorescence Microscopy
1.3 Proliferation and Cell Cycle Quantification by Flow Cytometry
1.4 Clonal Outgrowth to Measure Trypanocidal Activity
1.5 Purification of Intracellular Amastigotes
2 Materials
3 Methods
3.1 Preparation of T. cruzi Trypomastigotes for Use in In Vitro Infections
3.2 Multiplexed Assay to Measure Intracellular Parasite and Host Cell Abundance
3.3 Enumeration of Amastigote Growth by Microscopy
3.4 Flow Cytometry-Based Detection of Amastigote Proliferation
3.5 Quantification of Lethal Versus Static Effects on Amastigotes Through Clonal Outgrowth
3.6 PD-10 Column Purification of Intracellular T. cruzi Amastigotes
4 Notes
References
Chapter 33: Isolation of Extracellular Vesicles from Leishmania spp.
1 Introduction
2 Materials
2.1 Parasite Culture, Cleaning of Parasite Conditioned Medium
2.2 Ultracentrifugation
2.3 Polyethylene Glycol-Based Method (ExtraPEG)
2.4 Size-Exclusion Chromatography
2.5 Sucrose-Gradient Purification
2.6 Micro BCA Protein Assay
2.7 Nanoparticle Tracking Analysis
2.8 Negative Staining for TEM
2.9 Trichloroacetic Acid (TCA) Protein Precipitation
3 Methods
3.1 Cell Culture, Cleaning of Parasite Conditioned Medium
3.2 Ultracentrifugation
3.3 Polyethylene Glycol-Based Method (ExtraPEG)
3.4 Size-Exclusion Chromatography
3.5 Sucrose-Gradient Purification
3.6 Micro BCA Protein Assay
3.7 Nanoparticle Tracking Analysis
3.8 Negative Staining for TEM
3.9 TCA Protein Precipitation
4 Notes
References
Part V: Biochemistry
Chapter 34: 2D Gel Electrophoresis Analysis of Leishmania Proteomes
1 Introduction
2 Materials
3 Methods
3.1 Protein Extraction From Leishmania Promastigotes for 2D Gel Electrophoresis
3.1.1 Wash Cells
3.1.2 Lyse Cells
3.1.3 Protein Precipitation
3.1.4 Determine and Adjust Protein Content
3.2 Isoelectric Focussing (IEF)
3.2.1 Load Protein Sample Into IPG Gel Strip
3.2.2 Isoelectric Focusing of Protein Mixture
3.3 SDS-PAGE
3.3.1 IPG Strip Equilibration
3.3.2 SDS-PAGE Gel Preparation
3.3.3 SDS-PAGE Electrophoresis
3.3.4 Colloidal Coomassie Blue staining (See Note 7)
4 Notes
References
Chapter 35: Analysis of the Physiological and Metabolic State of Leishmania Using Heavy Water Labeling
1 Introduction
2 Materials
2.1 Parasite Cultivation In Vitro, 2H2O Labeling, and Harvest
2.2 The Infection, In Vivo 2H2O Labeling, Parasite Harvest, and Purification
2.3 Chloroform/Methanol/Water Extraction
2.4 Macromolecule Hydrolysis and Derivatization
2.4.1 DNA and RNA Hydrolysis and Derivatization
2.4.2 Protein Hydrolysis and Derivatization
2.4.3 Mannogen (Oligosaccharide) Hydrolysis and Derivatization
2.4.4 Lipid Hydrolysis and Derivatization
2.5 GC-MS Measurement and Data Analysis
3 Methods
3.1 Parasite Cultivation In Vitro, 2H2O Labeling, and Harvest
3.2 Infection, In Vivo 2H2O Labeling, Parasite Harvest, and Purification
3.3 Macromolecule Extraction
3.4 Macromolecule Hydrolysis and Derivatization
3.4.1 DNA and RNA Hydrolysis and Derivatization
3.4.2 Protein Hydrolysis and Derivatization
3.4.3 Mannogen Hydrolysis and Derivatization
3.4.4 Lipid Hydrolysis and Derivatization
3.5 GC-MS Measurement and Data Analysis
4 Notes
References
Chapter 36: A Scalable Purification Method for Mitochondria from Trypanosoma brucei
1 Introduction
2 Materials
2.1 Cell Growth and Harvest
2.2 Breakage of Cells, DNase I Digestion, Low-Speed Centrifugation
2.3 Density Gradient Centrifugation
2.4 Removal of Nycodenz and Storage
3 Methods
3.1 Cell Growth and Harvest
3.2 Breakage of Cells
3.3 DNase I Digestion
3.4 Low-Speed Centrifugation
3.5 Density Gradient Centrifugation
3.6 Removal of Nycodenz and Storage
4 Notes
Materials: Cell Growth and Harvest (See Subheading 2.1)
Methods
4.0.1 Methods: Cell Growth and Harvest (See Subheading 3.1)
4.0.2 Methods: Breakage of Cells (See Subheading 3.2)
4.0.3 Methods: DNase I Digestion (See Subheading 3.3)
4.0.4 Methods: Low-Speed Centrifugation (See Subheading 3.4)
4.0.5 Methods: Density Gradient Centrifugation (See Subheading 3.5)
4.0.6 Methods: Removal of Nycodenz (See Subheading 3.6)
References
Chapter 37: Isolation of Glycosomes from Trypanosoma cruzi
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Materials and Standard Equipment
2.3 Reagents and Buffer Solutions for Cell Rupture and Gradient Centrifugation
2.4 Solution of Sucrose for the Isopycnic Gradient
2.5 Cocktail of Protease Inhibitors
2.6 Silicon Carbide
2.7 Marker Enzyme Assay Reagents
3 Methods
3.1 Rupture of Parasites by Abrasion with Silicon Carbide
3.2 Preparation of an Enriched Fraction of Glycosomes by Differential Centrifugation
3.3 Preparation of Pure Glycosomes by Isopycnic Centrifugation
3.3.1 Making Gradients for Isopycnic Centrifugation
3.3.2 Fractionating Gradients
3.3.3 Location of Isolated Compartments Using Marker Enzymes
3.4 Determination of Intact Glycosomes: Latency
4 Notes
References
Chapter 38: Sorting the Muck from the Brass: Analysis of Protein Complexes and Cell Lysates
1 Introduction
2 Materials
2.1 SDS-Polyacrylamide Gel
2.2 Data Processing and Analysis
3 Methods
3.1 SDS Polyacrylamide Gel Electrophoresis
3.2 In-Gel Tryptic Digest and Mass Spectrometry
3.3 Data Processing and Label Free Quantification
3.4 Data Analysis
3.5 Validation
4 Notes
References
Chapter 39: Measurement of Energy States of the Trypanosomatid Mitochondrion
1 1 Introduction
2 Materials
2.1 Cells and Culture Conditions
2.2 Equipment
2.3 Reagents and Solutions
3 Methods
3.1 Preparation of Cell Samples and the OROBOROS for HRR
3.2 Measurements of HRR: Routine Respiration, Leak Respiration, ETS Capacity, and Residual Respiration
3.2.1 Routine Respiration
3.2.2 Leak Respiration
3.2.3 Electron Transfer Capacity
3.2.4 Residual Respiration Measurement
3.3 ATP Measurements
3.3.1 Kd Determination of ATP and ADP for Mg2+
3.3.2 ATP Net Production Measurement
3.3.3 ATP Assessment (Luciferase-Based Assay)
3.3.4 ATP Calibration Curve
3.4 ROS Measurement
3.4.1 H2O2 Calibration
3.4.2 Measurement of ROS Production Triggered by Omy
3.5 Mitochondrial Inner Membrane Potential (DeltaPsim) Measurement (Rhodamine 123 Based Assay)
4 Notes
References
Chapter 40: Isolation and Characterization of Acidocalcisomes from Trypanosomatids
1 Introduction
2 Materials
2.1 Cell Culture and Standard Equipment
2.2 Reagents
2.2.1 Gradient Centrifugations
2.2.2 Electron Microscopy
2.2.3 Marker Enzyme Assays
2.2.4 Immunoblotting
3 Methods
3.1 First Gradient Centrifugation
3.2 Second Gradient Centrifugation
3.3 Electron Microscopy
3.4 Marker Enzyme Assays
3.4.1 AMDP-Sensitive Vacuolar Proton Pyrophosphatase (V-PPase)
3.4.2 Succinate-Cytochrome c Reductase
3.4.3 Hexokinase
3.4.4 Alpha-Mannosidase
3.5 Western Blot Analyses
4 Notes
References
Chapter 41: Metabolic Control Analysis for Drug Target Prioritization in Trypanosomatids
1 Introduction
1.1 Determination of by In Vitro Pathway Reconstitution
1.1.1 Example: T(SH)2-Dependent Hydroperoxide Reduction Pathway from T. cruzi
1.2 Determination of by Manipulation of Enzyme Activities in Parasites
1.2.1 Example: Ex vivo T(SH)2 Synthesis Pathway Flux Control Distribution
1.3 Determination of by Kinetic Modeling of Metabolic Pathways
1.3.1 Example: Kinetic Model of Trypanothione Metabolism in T. cruzi
2 Materials
2.1 Enzyme Activity of Recombinant Enzymes
2.2 Parasite Cell Extract Preparation
2.3 Enzyme Activity in Parasite Cell Extracts
2.4 Pathway Reconstitution
2.5 Parasite Transfection
2.6 Selection and Cloning of Transfected Cells
2.7 Parasite Cell Extract Preparation
2.8 Enzyme Activities in Parasites
2.9 Ex Vivo Flux
2.10 Kinetic Parameters
2.11 Vmax Value of Each Enzyme/Transporter in the Parasites
2.12 Type of Kinetic Mechanism for Each Enzyme/Transporter
2.13 Software for Model Construction and Simulation
2.14 Dataset of Metabolite Concentration in the Parasites
2.15 Dataset of Pathway Fluxes
3 Methods
3.1 Determination of by In Vitro Pathway Reconstitution
3.1.1 Determination of Recombinant Enzyme Activities
3.1.2 Recombinant TryR Activity Assay
3.1.3 Recombinant TXN Activity Assay
3.1.4 Recombinant TXNPx Activity Assay
3.2 Parasite Extracts Production
3.3 Determination of Activities in Parasite Extracts
3.3.1 TryR Activity Assay in Parasite Extracts
3.3.2 TXN Activity Assay in Parasite Extracts
3.3.3 TXNPx Activity Assay in Parasite Extracts
3.4 In Vitro Pathway Reconstitution for Determination
3.4.1 Determination Assay for TryR
3.4.2 Determination Assay for TXN
3.4.3 Determination Assay for TXNPx
3.5 Determination of the
3.5.1 Plotting and Calculation for TryR
3.5.2 Plotting and Calculation for TXN
3.5.3 Plotting and Calculation for TXNPx
3.6 Determination of by Manipulating the Enzyme Activities in Parasites
3.7 Parasite Transfection
3.8 Selection and Cloning of Transfected Cells
3.9 Parasite Extracts Production from Overexpressing Clones
3.10 Determination of Enzyme Activities in Cell Extracts of Overexpressing Parasites
3.10.1 γECS Activity Assay
3.10.2 GS Activity Assay
3.10.3 TryS Activity Assay
3.11 Ex Vivo T(SH)2 Synthesis Flux Determination
3.12 Determination of the
3.12.1 Plotting and Calculation for γECS
3.12.2 Plotting and Calculation for TryS
3.12.3 Calculation for GS
3.13 Determination of by Kinetic Modeling of Metabolic Pathways
3.13.1 Model Construction
4 Notes
References
Part VI: Biomarkers and Diagnosis
Chapter 42: Establishment of a Standardized Vaccine Protocol for the Analysis of Protective Immune Responses During Experiment...
1 Introduction
2 Materials
2.1 Vaccine Materials
2.2 Mice and Parasites
2.3 Antibody Readout Reagents
2.4 FACS Readout Reagents
3 Method
3.1 Vaccination and Challenge Protocol
3.2 Preparation of Purified Dead Parasites for Vaccination Control Groups
3.3 Vaccine Induced Antibody Titer Determination (ELISA)
3.4 Overall Spleen Immunopathology Assessment (FACS)
4 Notes
References
Chapter 43: Isolation of Trypanosoma brucei brucei Infection-Derived Splenic Marginal Zone B Cells Based on CD1dHigh/B220High ...
1 Introduction
2 Materials
2.1 Mice, Infection, and Single Cell Suspensions
2.2 MACS-Based MZ B Cell Pre-enrichment
2.3 CD1dHigh/B220High-Based MZ B Cell Isolation Using FACS
2.4 RNA Isolation and 1.2% Agarose Formaldehyde Denaturing Electrophoresis
3 Methods
3.1 Mice, Infection, And Single Cell Suspensions
3.2 MACS-Based MZ B Cell Pre-enrichment
3.3 FACS-Based MZ B Cell Isolation
3.4 RNA Isolation and 1.2% Agarose Formaldehyde Denaturing Electrophoresis
4 Notes
References
Chapter 44: Cellular Markers for the Identification of Chemoresistant Isolates in Leishmania
1 Introduction
2 Materials
2.1 Solutions
2.2 Parasites
2.2.1 Reference Species Certified by the World Health Organization (WHO)
2.2.2 Parasites Isolated from Patient Lesions
3 Methods
3.1 Parasite Culture and Estimation of Cell Density
3.2 Glucose Utilization Rate
3.3 Plasma Membrane Potential (DeltaPsip)
3.3.1 Calibration of Cell Density vs. Fluorescence (DeltaF)
3.3.2 Calibration of Extracellular and Intracellular Concentration of Bis-Oxonol vs. Deltaf
3.3.3 Experimental Protocol for the Evaluation of DeltaPsip
3.4 Conclusion
4 Notes
References
Part VII: Drug Discovery for Trypanosomatid-Borne Diseases
Chapter 45: In Vitro Image-Based Assay for Trypanosoma cruzi Intracellular Forms
1 Introduction
2 Materials
3 Methods
3.1 T. cruzi Differentiation
3.2 LLC-MK2 Infection
3.3 Intracellular Image-Based Assay
4 Notes
References
Chapter 46: In Vitro Drug Efficacy Testing Against Trypanosoma brucei
1 Introduction
2 Materials
2.1 Cultivation Medium for T. brucei
2.2 Test Compounds
2.3 Cell Lines
2.4 Equipment
3 Methods
3.1 Low Inoculation, Long Incubation Test (LILIT)
3.2 Wash-Out Assay
3.3 Isothermal Microcalorimetry
4 Notes
References
Chapter 47: In Vitro Growth Inhibition Assays of Leishmania spp.
1 Introduction
2 Materials
2.1 Materials for Standard In Vitro Culture of Macrophage (mφ) Host Cells
2.2 Materials for Harvesting Primary Cells from Mice
2.3 Additional Materials for Obtaining Infective Stages of Leishmania for Infection
2.3.1 For Metacyclic Promastigotes
2.3.2 For Spleen-Derived Amastigotes
2.4 Additional Materials for Intracellular Amastigote Susceptibility Assay
3 Methods
3.1 In Vitro Culture of Macrophage (mφ) Host Cells (See also Note 2)
3.1.1 MEM Culture Medium
3.1.2 Complete RPMI-1640 Culture Medium
3.1.3 THP-1 Cell Culture
3.1.4 Harvesting and Culture of Primary Peritoneal Exudate Cells (PEC)
3.1.5 Harvesting and Culture of Primary Bone-Marrow Derived Macrophages (BMM)
3.2 Obtaining Leishmania Stages for Infection
3.2.1 Metacyclic Promastigotes
3.2.2 Ex Vivo Spleen-Derived Amastigotes (Only for Adapted Strains)
3.3 Intracellular Amastigote Susceptibility Assay
4 Notes
References
Chapter 48: In Vivo Bioluminescence Imaging to Assess Compound Efficacy Against Trypanosoma brucei
1 Introduction
2 Materials
2.1 Generation and Testing of Bioluminescent T. brucei Stabilates
2.1.1 Culturing of T. b. brucei GVR35 Bloodstream form Trypanosomes
2.1.2 Generation of Bioluminescent T. brucei Stabilates
2.1.3 Testing of Bioluminescent T. brucei Stabilates
2.2 In Vivo Imaging Model
2.2.1 Infection of Donor and Experimental Mice
2.2.2 Bioluminescence Imaging
2.2.3 Determine Blood Parasitemia
2.2.4 In Vivo Compound Treatments
2.2.5 Blood Sampling for Pharmacokinetic Analysis
2.2.6 End Point Imaging
3 Methods
3.1 Generation and Testing of Bioluminescent T. brucei Stabilates
3.1.1 Culturing of T. b. brucei GVR35-VSL2 Bloodstream form Trypanosomes
3.1.2 Generation of Bioluminescent T. brucei Blood Stabilates
3.1.3 Testing of Bioluminescent T. brucei Stabilates
3.2 In Vivo Imaging Model
3.2.1 Infection of Donor and Experimental Mice
3.2.2 Bioluminescence Imaging (See Note 4)
3.2.3 Determination of Blood Parasitemia
3.2.4 In Vivo Compound Treatments
3.2.5 Blood Sampling for Pharmacokinetic Analysis
3.2.6 End Point Imaging
3.3 Image Analysis
4 Notes
References
Index