Preface
References
Contents
Contributors
Part I: Biofilms and Subcellular Compartments
Chapter 1: Measuring Niche-Associated Metabolic Activity in Planktonic and Biofilm Bacteria
1 Introduction
2 Materials
2.1 Growth of Bacterial Strains and Preparation of Bacterial Stocks
2.2 Preparation of the Epithelial Substratum
2.3 Biofilm Formation In Vitro on Fixed Epithelial Cells
2.4 Biomass and Antibiotic Resistance of Planktonic and In Vitro Biofilm Bacteria
2.5 Scanning Electron Microscopy
2.6 Biofilm Dispersal with Heat In Vitro
2.7 Bacterial Energy Production
2.7.1 Oxidation Assay (Iodonitrotetrazolium, INT)
2.7.2 Determination of Intracellular ATP
2.8 Bacterial Fermentation Products
2.8.1 Lactate
2.8.2 Hydrogen Peroxide
2.8.3 Acetate
2.8.4 Ethanol
2.8.5 Formate
3 Methods
3.1 Adaptation and Growth of Planktonic Bacteria
3.1.1 Preparation of Planktonic Bacterial Strains
3.1.2 Preparation of the Epithelial Substratum
3.2 Biofilm Formation In Vitro
3.3 Bacterial Density and Antibiotic Resistance of Planktonic and Biofilm Bacteria
3.4 Assessment of Biofilm Structure by Scanning Electron Microscopy (SEM)
3.5 Biofilm Heat Dispersal In Vitro
3.6 Overall Energy Metabolism
3.6.1 Oxidation Assay
3.6.2 ATP Production
3.7 Measuring Fermentation Metabolites
3.7.1 Inducing Bacterial Metabolism and Fermentation
3.7.2 Lactate Determination
3.7.3 Hydrogen Peroxide Determination
3.7.4 Acetate Determination
3.7.5 Ethanol Determination
3.7.6 Formate Determination
4 Notes
References
Chapter 2: Formation and Analysis of Mono-species and Polymicrobial Oral Biofilms in Flow-Cell Models
1 Introduction
2 Materials
2.1 Coating of Flow-Cell Surfaces
2.1.1 Bacteria-Free Saliva or Salivary Fractions
2.1.2 Serum
2.2 Fluorescence in Situ Hybridization (FISH)
2.3 Glycosidase Activity
2.4 Analysis of Biofilm Acid Tolerance
2.5 Extraction of Intracellular Proteins
2.6 Two-Dimensional Polyacrylamide Gel Electrophoresis (2DE)
3 Methods
3.1 Preparation of Bacterial Cultures for Biofilm Experiments
3.1.1 Preparation of Bacteria for Mono-species Experiments
3.1.2 Preparation of Bacteria for Multi-species Biofilm Experiments
3.2 Coating of Flow-Cell Surfaces
3.3 Biofilm Models
3.3.1 Static Biofilm Models
3.3.2 Flow-Cell Models
3.4 Biofilm Analysis
3.4.1 Analysis of Biofilm Mass In Situ
3.4.2 Analysis of Surface Coverage and Bacterial Viability in Biofilms In Situ
3.4.3 Analysis of Gram-Positive and Gram-Negative Bacteria in Biofilms In Situ
3.4.4 Analysis of Biofilm Composition In Situ Using 16S r-RNA Fluorescent In Situ Hybridization (FISH)
3.4.5 Analysis of Metabolic Activity in Biofilms In Situ
3.4.6 Analysis of Biofilm Proteolytic Activity In Situ
3.4.7 Analysis of Biofilm Acid Tolerance In Situ
3.4.8 Analysis of Composition of Bacteria Recovered from Biofilms
3.4.9 Analysis of Glycosidase Activity in Bacteria Recovered from Biofilms
3.4.10 Analysis of Lactic Acid from Bacteria Recovered from Biofilms
3.4.11 Analysis of Acetic Acid from Bacteria Recovered from Biofilms
3.5 Extraction of Intracellular Proteins from Biofilm Bacteria
3.6 Analyses of Intracellular Protein Profiles of Biofilm Bacteria
3.6.1 Isoelectric Focusing (IEF)
3.6.2 Equilibration of IPG Strips
3.6.3 Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.6.4 Visualization and Identification of Proteins Following 2DE
4 Notes
References
Chapter 3: Isolation and Purification of Mycobacterial Extracellular Vesicles (EVs)
1 Introduction
2 Materials
2.1 Vesicle Induction
2.2 Vesicle Harvesting
2.3 Vesicle Purification
3 Methods
3.1 Vesicle Induction
3.2 Vesicle Harvesting
3.3 Vesicle Purification
4 Notes
References
Chapter 4: Strategies to Isolate Extracellular Vesicles from Gram-Negative and Gram-Positive Bacteria
1 Introduction
2 Materials
2.1 Bacterial Culture Media
2.2 Supernatant Containing Membrane Vesicle Preparation
2.3 Ultracentrifugation
2.4 Discontinuous Iodixanol Gradient Separation
2.5 SDS-PAGE
3 Methods
3.1 Cultivation of S. pneumoniae and K. pneumoniae
3.2 Isolation of Membrane Vesicles from Supernatant Cultures (see Note 3)
3.3 Membrane Vesicle Purification Through OptiPrep Density Gradient
4 Notes
References
Part II: Bacterial Genetics, Genomics, and Phylogenetics
Chapter 5: Total Bacterial RNA Isolation and Northern Blotting Analysis
1 Introduction
2 Materials
2.1 RNA Isolation using TRIzol-Acid Phenol
2.2 RNA Isolation Using Hot Phenol
2.3 Determining the Quality and Quantity of RNA
2.4 DNase Treatment
2.5 Northern Blot
2.6 Probe Preparation
2.7 Membrane Exposure and Development
3 Methods
3.1 RNA Isolation Using TRIzol-Acid Phenol
3.2 RNA Isolation Using Hot Phenol
3.3 Determining the Quality and Quantity of RNA
3.3.1 Agarose Gel Method
3.3.2 Nucleic Acid Spectrophotometer Method
3.4 DNase Treatment (see Note 8)
3.5 Northern Blot
3.5.1 RNA Sample Preparation and Formaldehyde Agarose Gel
3.5.2 Transfer of RNA from Gel onto Membrane
3.5.3 Hybridization of Membrane and Probe Preparation
3.6 Probe Preparations
3.6.1 End-Labeling with Polynucleotide Kinase and Isotope ฮณ-dATP
3.6.2 Random Primer Labeling with Isotope ฮฑ-dATP (see Note 15)
3.7 Membrane Exposure and Development
4 Notes
References
Chapter 6: Phylogenetic Analysis of Bacterial Pathogen Genomes
1 Introduction
2 Materials
2.1 Genomic Data
2.2 Computer Hardware
2.3 Computer Software
3 Methods
3.1 Producing the Input Alignment
3.2 Building an Initial Phylogeny
3.3 Recombination Analysis
3.4 File Outputs
3.5 Graphical Representation
3.6 Downstream Analyses
4 Notes
References
Chapter 7: Determination of Growth Rate and Virulence Plasmid Copy Number During Yersinia pseudotuberculosis Infection Using D...
1 Introduction
2 Materials
2.1 DNA Extraction and Quantification
2.2 ddPCR Supermix Components
2.3 Droplet Generation and Droplet Reader Components
3 Methods
3.1 Sample Acquisition
3.2 DNA Extraction
3.3 Primer Design
3.4 Preparation of Reaction Mixtures
3.5 Droplet Generation
3.6 PCR Reaction
3.7 Droplet Reading
3.8 Data Analysis
3.9 Statistical Errors
4 Notes
References
Part III: Identification and Characterization of Bacterial Effector Proteins
Chapter 8: Methods to Analyze the Contribution of Complement Evasion Factor (CEF) to Streptococcus pyogenes Virulence
1 Introduction
2 Materials
2.1 Binding of CEF to Human Complement Proteins
2.2 Complement Hemolytic Assay
2.3 Complement Pathway Interference Assay
2.4 Complement Deposition Assay
3 Methods
3.1 Binding of CEF to Human Complement Proteins
3.2 Complement Hemolytic Assay (Fig. 1)
3.3 Complement Interference Assay
3.4 Complement Deposition Assay (Fig. 2)
4 Notes
References
Chapter 9: Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae
1 Introduction
2 Materials
2.1 Transformation of Yeast Cells
2.2 Characterization
2.2.1 Cultivations
2.2.2 Cell Lysis
Y-PER
Bead Beater
2.2.3 Western Blot
2.2.4 Activity Assay
2.3 Flow Cytometry
3 Methods
3.1 Construct Design
3.2 Transformation of Yeast Cells
3.2.1 Day 1
3.2.2 Day 2
3.3 Characterization of Yeast Strains
3.3.1 Cultivations
3.3.2 Cell Lysis
Y-PER
Bead Beater
3.3.3 Western Blot
3.3.4 Activity Assay
3.4 Flow Cytometry Analysis
3.4.1 Flow Cytometry Data
4 Notes
References
Chapter 10: Mass Spectrometry-Based Methods to Determine the Substrate Specificities and Kinetics of N-Linked Glycan Hydrolysi...
1 Introduction
1.1 Endo-ฮฒ-N-Acetylglucosaminidases
1.2 Chemoenzymatic Remodeling of Glycoproteins
2 SEAK/C-SEAK Protocol
2.1 Materials
2.1.1 Bacterial Strains and Media
2.1.2 Eukaryotic Expression Strains
2.1.3 Plasmids and Cloning
2.1.4 Proteins
2.1.5 Protein Purification
2.1.6 Materials for Eukaryotic Expression
2.1.7 Mass Spectrometry Materials
2.1.8 Instruments
2.1.9 Software
3 Methods
3.1 Cloning to Produce Fc Plasmid
3.2 Expression and Purification of EndoS/EndoS2
3.3 Transfection and Expression of HM-Rituximab and Fc
3.4 Purification of IgG Antibodies and Fc
3.5 LC-MS Setup for Glycoprotein Analysis
3.6 SEAK with EndoS
3.7 SEAK with EndoBT-3987
3.8 C-SEAK with EndoS2
3.9 C-SEAK with EndoBT-3987
3.10 Importing Data into Excel
3.11 Exporting Data from Excel into KinTek
4 Measuring ENGase Activities and Specificities
4.1 Chromatographic Methods Combined with Densitometry Analysis
4.1.1 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
4.1.2 Thin-Layer Chromatography (TLC)
4.1.3 Capillary Electrophoresis
4.2 Spectroscopic Methods
4.2.1 UV and Fluorescence Spectroscopy
4.2.2 Mass Spectrometry-Based Approaches
Bottom-Up Approaches
Intact Mass Spectrometry Approaches
Competitive Universal Proxy Receptor Assay (CUPRA-ZYME)
SEAK and C-SEAK
5 Notes
References
Chapter 11: Identification of Substrates of Secreted Bacterial Protease by APEX2-Based Proximity Labeling
1 Introduction
2 Materials
2.1 APEX2-Based Proximity Labeling
2.2 Immunofluorescence and Western Blot
2.3 Biotinylated Proteins Enrichment and On-Beads Digestion
2.4 TMT Peptide Labeling and Cleaning-Up
2.5 Mass Spectrometry Analysis
3 Methods
3.1 APEX2-Based Proximity Labeling of Live Cells
3.1.1 Purification of APEX2-Fusion Protein
3.1.2 Proximity Labeling of Live Cells
3.2 Immunofluorescence and Western Blot Analysis of Labeled Cells
3.2.1 Immunofluorescence Analysis
3.2.2 Western Blot Analysis
3.3 Biotinylated Proteins Enrichment and On-Beads Trypsin Digestion
3.4 TMT Peptide Labeling and Cleaning-Up
3.5 Mass Spectrometry Analysis
4 Notes
References
Chapter 12: Affinity-Purification Combined with Crosslinking Mass Spectrometry for Identification and Structural Modeling of H...
1 Introduction
2 Materials
2.1 Affinity-Purification
2.2 Crosslinking
2.3 SDS-PAGE Analysis
2.4 Sample Precipitation and In-Solution Digestion for Mass Spectrometry
2.5 Peptide C18 Solid Phase Extraction
2.6 Mass Spectrometry
2.7 Software for Data Analysis, Statistic Evaluation, Structural Docking, and Visualization
3 Methods
3.1 Affinity-Purification
3.2 Crosslinking
3.3 SDS-PAGE
3.4 Sample Precipitation and In-Solution Digestion for Mass Spectrometry
3.5 Peptide C18 Solid Phase Extraction
3.6 Mass Spectrometry
3.7 Quantitative Mass Spectrometry Data Analysis to Identify Interacting Proteins
4 Crosslinking Mass Spectrometry Data Analysis
4.1 Visualizing the Crosslinked Interfaces
4.2 Docking Selected Proteins Together and Visualizing the Complex(es)
5 Notes
References
Chapter 13: Elucidating the Stoichiometries of Host-Pathogen Protein Interactions with Mass Photometry
1 Introduction
2 Materials
3 Methods
4 Notes
References
Part IV: Analysis of Host Responses to Bacteria
Chapter 14: Analysis of Neutrophil and Monocyte Inflammation Markers in Response to Gram-Positive Anaerobic Cocci
1 Introduction
2 Materials
2.1 Bacteria, Medium, Lab Consumables, and Kits
2.2 Antibodies and Dilutions
3 Methods
3.1 Growth of Bacteria
3.2 Interaction of Whole Blood and P. harei Strain 5984
3.3 Enzyme-Linked Immunosorbent Assay (ELISA)
3.4 Flow Cytometry
3.4.1 Surface Markers
3.4.2 Intracellular Cytokines
4 Notes
References
Chapter 15: Quantification of Phagocytosis Using Flow Cytometry
1 Introduction
2 Materials
2.1 Prey Preparation
2.2 Phagocyte Preparation
2.3 Fluorescent Dyes and Flow Cytometer
3 Methods
3.1 Bacterial Culture and Heat-Killing
3.2 Staining of Bacteria
3.3 Sonicate, Count Prey, and Check Staining
3.4 Opsonize
3.5 Prepare Plate (See Note 12)
3.6 Culture Phagocytes and Count
3.7 Phagocytosis and Ice Controls
3.8 Post-Acquisition Analysis (See Note 19)
3.8.1 Curve Analysis (See Note 20)
3.8.2 Population Assessment
3.8.3 Individual Phagocyte Assessment
3.8.4 PAN-Analysis to Determine MOP50, Hill Coefficient, and Top Value
3.8.5 PAN-Analysis to Determine Prey Per Phagocyte, PxP
4 Notes
References
Chapter 16: Antibacterial Neutrophil Effector Response: Ex Vivo Quantification of Regulated Cell Death Associated with Extrace...
1 Introduction
2 Materials
2.1 Neutrophil Isolation
2.2 Preparation of Bacteria
2.3 Regulated Cell Death Kinetics Analysis by Flow Cytometry
2.4 NETs Release Kinetics Analysis by Flow Cytometry
2.5 Cell Death and NETs Release Analysis by Time-Lapse Microscopy
3 Methods
3.1 Neutrophil Isolation
3.2 Bacteria Preparation and Infection
3.3 Regulated Cell Death Kinetics Analysis by Flow Cytometry
3.4 NETs Release Kinetics Analysis by Flow Cytometry
3.5 Cell Death and NETs Release Analysis by Live-Imaging Microscopy
4 Notes
References
Chapter 17: Measurement of Antibody Binding Affinity on Bacterial Surfaces Using Flow Cytometry
1 Introduction
2 Material
2.1 Bacteria and Antibody Reagents
2.2 Fluorescent Reagents
3 Method
3.1 Prepare Bacteria
3.2 Prepare Antibodies
3.3 Prepare Samples
3.4 Flow Cytometry
3.5 Affinity Calculation
4 Notes
References
Chapter 18: Detection of Inflammasome Activation in Murine Bone Marrow-Derived Macrophages Infected with Group A Streptococcus
1 Introduction
2 Materials
2.1 Harvesting Bone Marrow and Generating Bone Marrow-Derived Macrophages (BMDMs)
2.1.1 Femur Excision
2.1.2 Bone Marrow (BM) Harvest
2.1.3 Generation of BMDMs from Bone Marrow
2.1.4 Thawing of Frozen BMDMs
2.2 Bacterial Culture
2.2.1 Streaking GAS on Blood Agar (Can be done up to a week before infection)
2.2.2 Inoculation of an Overnight Culture (The day before infection)
2.3 Infection of BMDMs with GAS
2.3.1 Re-plate BMDMs onto Non-tissue Culture-Treated Plates (The day before infection)
2.3.2 Prime the BMDMs with LPS (4-15 h before infection)
2.3.3 Infect the BMDMs with GAS
2.3.4 Serial Dilution for MOI Determination
2.4 Inflammasome Activation Readouts (See Note 9)
2.4.1 Measure Release of Secreted Factors
2.4.2 Detect Mature Cytokine and Active Caspase-1 by Western Blot
2.4.3 Measure In-Cell Caspase-1 Activation
2.4.4 Observe and Measure Cell Death Induction
Observe Cell Death Induction
Measure Cell Death by LDH Release
Measure Cell Death by PI Uptake
3 Methods
3.1 Harvesting Bone Marrow and Generating BMDMs
3.1.1 Femur Excision
3.1.2 Bone Marrow Harvest
3.1.3 Generation of BMDMs from Bone Marrow
3.1.4 Thawing of Frozen BMDMs (See Note 31)
3.2 Bacterial Culture
3.2.1 Streaking GAS on Blood Agar
3.2.2 Inoculate an Overnight Culture
3.3 Infection of BMDMs with GAS
3.3.1 Re-plate BMDMs onto Non-tissue Culture-Treated Plates
3.3.2 Prime the BMDMs with LPS
3.3.3 Infect the BMDMs with GAS
3.3.4 Serial Dilution for MOI Determination
3.4 Inflammasome Activation Readouts (See Notes 46 and 47)
3.4.1 Measure Release of Secreted Factors
3.4.2 Detect Mature Cytokines and Active Caspase-1 by Western Blot
3.4.3 Measure In-Cell Caspase-1 Activation
3.4.4 Observe and Measure Cell Death Induction
Observe Morphology of Cells
Measure Cell Death by LDH Release
Measure Cell Death by PI Uptake
4 Notes
4.1 Materials
4.2 Methods
References
Part V: In Vivo and In Vitro Infection Models
Chapter 19: In Vivo Profiling of the Vascular Cell Surface Proteome in Murine Models of Bacteremia
1 Introduction
2 Materials
2.1 Bacterial Infection
2.2 In Vivo Chemical Perfusions
2.3 Automated Streptavidin Enrichment, Trypsinization, and Peptide C18 Clean-up
2.4 Shotgun Proteomics Analysis
2.5 Data Analysis
3 Methods
3.1 Bacterial Infection
3.2 In Vivo Perfusions and Organ Homogenization
3.3 Automated Streptavidin Enrichment and Trypsinization
3.4 Automated Peptide C18 Clean-up
3.5 Shotgun Proteomics Analysis
3.6 Data Analysis Using MaxQuant and Perseus
3.7 Expected Results
4 Notes
References
Chapter 20: Barcoded Consortium Infections: A Scalable, Internally Controlled Method to Study Host Cell Binding and Invasion b...
1 Introduction
2 Materials
2.1 Bacterial Strain Construction and Culturing
2.2 Cell Line Culture and Infections
2.3 Animal Infections
2.4 gDNA Extraction and qPCR Quantification
3 Methods
3.1 Tagging of Salmonella Mutants with Neutral Genetic Tags
3.2 Barcoded Consortium Infections of Cell Lines
3.2.1 Infections of Epithelial Cell Lines
3.2.2 Infections of Monocyte/Macrophage Cell Lines
3.2.3 Barcoded Consortium Host Cell Binding Assays
3.3 Barcoded Consortium Infections in a Murine Gut Infection Model
3.4 Quantification of Tagged Strain Abundances by qPCR
4 Notes
References
Chapter 21: A Murine Mycobacterium marinum Infection Model for Longitudinal Analyses of Disease Development and the Inflammato...
1 Introduction
2 Materials
2.1 Preparation of M. marinum for Infection
2.2 Intravenous Injection Via the Tail Vein
2.3 CFU Enumeration from the Infected Tail Tissue
2.4 Preparation of Tail Tissue for Flow Cytometry Analyses
2.5 Preparation of Tail Tissue for Immunofluorescence Microscopy
2.6 Preparation of Tail Skin Tissue for Protein Content Analyses or RNA Extraction
3 Methods
3.1 Mouse Infection Model
3.1.1 Preparation of M. marinum for Infection
3.1.2 Intravenous Injection Via the Tail Vein
3.1.3 Monitoring of Disease Development: Quantification of Visible Tail Lesions
3.1.4 Analysis of Bacterial Growth in the Infected Tail Tissue by CFU Enumeration
3.1.5 Preparation of Tail Tissue for Downstream Flow Cytometry Analyses
3.1.6 Preparation of Tails for Immunofluorescence Microscopy
3.1.7 Preparation of Tail Skin Tissue for Cytokines and Protein Content Analyses
3.1.8 Preparation of Tail Skin Tissue for RNA Extraction
4 Notes
References
Chapter 22: In Vitro Approaches for the Study of Pneumococcal-Neuronal Interaction and Pathogenesis
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Infection
2.3 Immunocytochemistry
3 Methods
3.1 Differentiation of Neurons
3.2 Infection
3.3 Adhesion
3.4 Invasion
3.5 Immunocytochemistry
4 Notes
References
Index