Periodontal Pathogens: Methods and Protocols

Preface
Contents
Contributors
Part I: Methods for Bacterial Genetic Manipulation
	Chapter 1: Site-Directed and Random Mutagenesis in Porphyromonas gingivalis: Construction of Fimbriae-Related-Gene Mutant
		1 Introduction
		2 Materials
			2.1 Site-Directed Mutagenesis
				2.1.1 Culture Media and Antibiotics for Escherichia coli and P. gingivalis
				2.1.2 Preparation of DNA Constructs
				2.1.3 Preparation of P. gingivalis Competent Cells
				2.1.4 Electroporation of P. gingivalis Cells
			2.2 Random Mutagenesis
				2.2.1 Transposon Mutagenesis of P. gingivalis by Conjugation
				2.2.2 Colony Immunoblotting
		3 Methods
			3.1 Site-Directed Mutagenesis
				3.1.1 Culture Conditions for P. gingivalis and E. coli
				3.1.2 Preparation of DNA Constructs
				3.1.3 Competent Cell Preparation of P. gingivalis
				3.1.4 Electroporation of P. gingivalis
			3.2 Random Mutagenesis
				3.2.1 Transposon Mutagenesis of P. gingivalis by Conjugation
				3.2.2 Screening of Mutants (e.g., Lacking Outer Membrane Proteins): Colony Immunoblotting
		4 Notes
		References
	Chapter 2: Genetic Manipulations of Oral Spirochete Treponema denticola
		1 Introduction
		2 Materials
		3 Methods
			3.1 Designing Constructs for Gene Deletion by Allelic Exchange
			3.2 Designing Construct for Gene Deletion by Counterselectable Maker
			3.3 Preparing Electrocompetent Cells
			3.4 Electrotransformation
			3.5 Preparing Chemically Induced Competent Cells
			3.6 Heat Shock Transformation
			3.7 Selection of Transformants by Plating
			3.8 Cis-Complementation via Genetic Reconstitution
			3.9 Trans-Complementation Using a Shuttle Vector (See Note 9)
		4 Notes
		References
	Chapter 3: Construction of a Gene-Deletion Mutant in Tannerella forsythia
		1 Introduction
		2 Materials
			2.1 Bacterial Culture
			2.2 Inactivation of the Target  Gene
		3 Methods
			3.1 Blood Agar Medium with  NAM
			3.2 Preparation of Electrocompetent Cells of T. forsythia
			3.3 Preparation of DNA Construct for Allelic Exchange Mutagenesis
			3.4 Transformation
		4 Notes
		References
	Chapter 4: Construction of a Mutant in Prevotella melaninogenica Using the Conjugation Transfer Method with Escherichia coli
		1 Introduction
		2 Materials
			2.1 A porK-Deletion Mutant of P. melaninogenica
			2.2 Hemagglutination Test
		3 Methods
			3.1 Construction of a porK-Deletion Mutant of P. melaninogenica
				3.1.1 Construction of Suicide Vector Plasmid
				3.1.2 Introduce the Suicide Vector Plasmid into E. coli
				3.1.3 Conjugative Transfer
				3.1.4 Confirm that the Target Gene Has Been Replaced with ermF
			3.2 Hemagglutination  Test
		4 Notes
		References
	Chapter 5: Genetic Transformation of Fusobacterium nucleatum
		1 Introduction
		2 Materials
			2.1 Plasmid DNA Construct for Transformation
			2.2 Transformation of F. nucleatum
				2.2.1 Bacterial Preparation
				2.2.2 Sonoporation
		3 Methods
			3.1 Construction of a fadA-Deletion Mutant in F. nucleatum
				3.1.1 DNA Construct
				3.1.2 Transformation Using Sonoporation
			3.2 Construction of a fadA-Complemented Mutant in F. nucleatum
				3.2.1 DNA Construct
				3.2.2 Transformation Using Sonoporation
		4 Notes
		References
Part II: Experimental Methods to Examine Virulence Factors
	Chapter 6: Genotyping of Porphyromonas gingivalis in Relationship to Virulence
		1 Introduction
		2 Materials
			2.1 Samples and P. gingivalis Culture
			2.2 PCR
			2.3 Electrophoresis
		3 Methods
			3.1 Clinical Sample Collection
			3.2 DNA Extraction
			3.3 Confirm Existence of Bacterial DNA Using  PCR
			3.4 Confirm Existence of P. gingivalis DNA Using  PCR
			3.5 Determine fimA Genotypes of P. gingivalis-Positive Specimens Using  PCR
			3.6 Second PCR for fimA Type-Unidentified Specimens Obtained in First PCR Assay (Nested PCR)
		4 Note
		References
	Chapter 7: Transport and Polymerization of Porphyromonas gingivalis Type V Pili
		1 Introduction
		2 Materials
			2.1 Bacterial Culture
			2.2 Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel
			2.3 Immunoblotting
			2.4 Globomycin Treatment
			2.5 Palmitic Acid Labeling Experiment
			2.6 Dot Blot Analysis
			2.7 Cys-Cys Cross-Linking Analysis
			2.8 Electron Micrography
			2.9 Preparation of Fim Fimbriae
		3 Methods
			3.1 Globomycin Treatment
			3.2 Palmitic Acid Labeling Experiment
			3.3 Dot Blot Analysis
			3.4 Cys-Cys Cross-Linking Analysis
			3.5 Electron Microscopy
		4 Notes
		References
	Chapter 8: Purification of Native Mfa1 Fimbriae from Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 Cultivation of P. gingivalis
			2.2 Preparation and Isolation of Fimbriae
				2.2.1 Preparation of the Soluble Fraction from P. gingivalis Cells
				2.2.2 Isolation and Purification of Fimbriae
			2.3 SDS-PAGE
			2.4 Immunoblotting
			2.5 Electron Microscopy
		3 Methods
			3.1 Preparation and Isolation of Fimbriae
				3.1.1 Culturing of P. gingivalis
				3.1.2 Preparation of the Soluble Fraction
				3.1.3 Isolation and Purification of Fimbriae
			3.2 Assays for the Purity of Fimbriae
				3.2.1 SDS-PAGE
				3.2.2 Immunoblotting
				3.2.3 Electron Microscopy
		4 Notes
		References
	Chapter 9: Crystallization of Recombinant Fimbrial Proteins of Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 Initial Screening
			2.2 Optimization
			2.3 Cryo Crystallography
		3 Methods
			3.1 Protein Crystallization
			3.2 Optimization
			3.3 Cryoprotection of Crystals
		4 Notes
		References
	Chapter 10: Enzymatic Characteristics and Activities of Gingipains from Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 Gingipain Sample Preparation
			2.2 Substrates for Rgp
			2.3 Substrates for Kgp
			2.4 Protein Substrates
			2.5 Measurement of Gingipain Activity Using MCA Substrates
			2.6 Measurement of Gingipain Activity Using p-Nitroanilide Substrates
			2.7 Measurement of Hemoglobin-Hydrolyzing Activity
			2.8 Measurement of Caseinolytic Activity
			2.9 Gelatin Zymography
			2.10 Luminol-Dependent Chemiluminescence (CL) Response of Neutrophils
			2.11 Measurement of Vascular Permeability In Vivo
		3 Methods
			3.1 Gingipain Sample Preparation
			3.2 Measurement of Gingipain Activity Using MCA Substrates
				3.2.1 Gingipain Activity in Sample
				3.2.2 Standard Curve
			3.3 Measurement of Gingipain Activity Using p-Nitroanilide Substrates
			3.4 Measurement of Hemoglobin-Hydrolyzing Activity
				3.4.1 Gingipain Activity in Sample
				3.4.2 Standard Curve
			3.5 Measurement of Caseinolytic Activity
				3.5.1 Gingipain Activity in Sample
				3.5.2 Standard Curve
			3.6 Gelatin Zymography
			3.7 Luminol-Dependent Chemiluminescence (CL) Response of Neutrophils
				3.7.1 Neutrophil Preparation
				3.7.2 Opsonization of Zymozan A
				3.7.3 Chemiluminescence Assay
			3.8 Measurement of Vascular Permeability In Vivo
		4 Notes
		References
	Chapter 11: Structural Characterization of the Type IX Secretion System in Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 PorK/N Complex Purification
			2.2 Electron Microscopy
			2.3 Cross-Linking
		3 Methods
			3.1 Isolation of PorK/N Protein Complex
			3.2 Electron Microscopy
				3.2.1 Negative Staining
				3.2.2 Cryo-Electron Microscopy
			3.3 Cross-Linking Mass Spectrometry
		4 Notes
		References
	Chapter 12: Methods for Functional Characterization of the Type IX Secretion System of Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 A T9SS-Deficient Mutant of P. gingivalis
			2.2 T9SS Complemented Strains of P. gingivalis
			2.3 Colony Pigmentation
			2.4 Analysis of Progingipains
			2.5 Hemagglutination Test
			2.6 Subcellular Fractionation
			2.7 Analysis of the Supernatant Protein
		3 Methods
			3.1 Construction of a T9SS Deficient Mutant of P. gingivalis
				3.1.1 Construction of Targeting Vector Plasmid
				3.1.2 Construction of a P. gingivalis Drug Resistant Mutant Strain
				3.1.3 Construction of a P. gingivalis Multidrug-Resistant Mutant Strain
			3.2 Construction of a T9SS Complemented Strain of P. gingivalis
				3.2.1 Construction of Targeting Vector Plasmid
				3.2.2 Introduction of the Targeting Vector Plasmid into E. coli
				3.2.3 Conjugative Transfer
			3.3 Colony Pigmentation
			3.4 Analysis of Progingipains
			3.5 Hemagglutination Test
			3.6 Subcellular Fractionation
			3.7 Analysis of the Supernatant Protein
		4 Notes
		References
	Chapter 13: Purification of Tannerella forsythia Surface-Layer (S-Layer) Proteins
		1 Introduction
		2 Materials
			2.1 T. forsythia Culturing
			2.2 Extraction and Partial Purification of T. forsythia Surface Layer
			2.3 Size Exclusion Chromatography
		3 Methods
			3.1 T. forsythia Culturing
			3.2 Extraction and Partial Purification of T. forsythia Surface Layer
			3.3 Size Exclusion Chromatography
		4 Notes
		References
	Chapter 14: Separation of Glycosylated OmpA-Like Proteins from Porphyromonas gingivalis and Tannerella forsythia
		1 Introduction
		2 Materials
			2.1 Growth of Bacterial Cells
			2.2 Preparation of Bacterial Whole-Cell Lysates
			2.3 Solubilization of Bacterial Whole-Cell Lysates
			2.4 Lectin Affinity Chromatography
			2.5 SDS-PAGE
			2.6 Detection of Proteins
			2.7 Detection of Glycosylated Proteins Using Glycoprotein Stain
			2.8 Western Blotting
				2.8.1 Confirmation of OmpA-Like Proteins Using Specific Antibody (SeeNote 9)
				2.8.2 Detection of O-GlcNAc Modification Using Anti-O-GlcNAc Antibody
				2.8.3 Confirmation of WGA Reactivity by Lectin Blotting
		3 Methods
			3.1 Growth of Anaerobic Bacterial Cells
			3.2 Preparation of Bacterial Whole-Cell Lysates
			3.3 Solubilization of Bacterial Whole-Cell Lysates
			3.4 Lectin Affinity Chromatography (SeeNote 12)
			3.5 SDS-PAGE
			3.6 Detection of Proteins
			3.7 Detection of Glycosylated Proteins (SeeNote 15)
			3.8 Western Blotting
				3.8.1 Detection of OmpA-Like Proteins
				3.8.2 Detection of O-GlcNAc Modification (SeeNote 16)
				3.8.3 Lectin Blotting
		4 Notes
		References
	Chapter 15: Intranasal Vaccine Study Using Porphyromonas gingivalis Membrane Vesicles: Isolation Method and Application to a M...
		1 Introduction
		2 Materials
			2.1 Cultivation of P. gingivalis for MV Preparation
			2.2 MV Preparation
			2.3 Purification of MVs by Density Gradient Centrifugation (See Note 3)
			2.4 Quantification of Lipids Contained in MVs (See Note 4)
			2.5 Intranasal Immunization of MVs to  Mice
		3 Methods
			3.1 MV Preparation and Quantification
				3.1.1 MV Preparation
				3.1.2 Quantification of Lipids Contained in  MVs
			3.2 Intranasal Immunization of MVs to  Mice
				3.2.1 Immunization
				3.2.2 Collection of Mouse Specimens and Antibody Detection
		4 Notes
		References
	Chapter 16: Analysis of the Butyrate-Producing Pathway in Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 Colorimetric Assay for Butyryl-CoA:Acetate CoA Transferase Activity
			2.2 GC-MS Assay for Detection of SCFAs
		3 Methods
			3.1 Colorimetric Assay for Butyryl-CoA:Acetate CoA Transferase
			3.2 GC-MS Analysis
		4 Notes
		References
	Chapter 17: Characterization of the Treponema denticola Virulence Factor Dentilisin
		1 Introduction
		2 Materials
			2.1 Purification of Dentilisin
			2.2 Measurement of Dentilisin Activity
			2.3 Evaluation of Pathogenicity of T. denticola
			2.4 Construction of T. denticola Dentilisin Mutant (Electroporation Protocol)
			2.5 Construction of T. denticola Dentilisin Mutant (Heat Shock Protocol)
			2.6 Coaggregation Assay
			2.7 Measurement of the Invasion Potential of T. denticola (Antibiotic Protection Assay)
		3 Methods
			3.1 Purification of Dentilisin
			3.2 Measurement of Dentilisin Activity
			3.3 Evaluation of Pathogenicity of T. denticola
			3.4 Construction of T. denticola Dentilisin Mutant (Electroporation Protocol)
				3.4.1 Construction of DNA Construct
				3.4.2 Preparation of Competent Cells
				3.4.3 Transformation of T. denticola
			3.5 Construction of T. denticola Mutant (Heat Shock Protocol)
				3.5.1 Preparation of Competent Cells
				3.5.2 Transformation of T. denticola
			3.6 Coaggregation Assay
			3.7 Measurement of the Invasion Potential of T. denticola (Antibiotic Protection Assay)
		4 Notes
		References
	Chapter 18: Evaluation of the Virulence of Aggregatibacter actinomycetemcomitans Through the Analysis of Leukotoxin
		1 Introduction
		2 Materials
			2.1 Determination of Leukotoxin Promoter Type
				2.1.1 Dental Plaque Preparation
				2.1.2 Polymerase Chain Reaction (PCR)
			2.2 Apoptosis Assay
				2.2.1 Infection of Leukocytes with A. actinomycetemcomitans
				2.2.2 DNA Laddering
				2.2.3 Annexin V Apoptosis Detection Assay
				2.2.4 Determination of LDH Release
		3 Methods
			3.1 Determination of Leukotoxin Promoter Type
				3.1.1 Dental Plaque Preparation
				3.1.2 PCR
			3.2 Apoptosis Assay
				3.2.1 Infection of THP-1 with A. actinomycetemcomitans
				3.2.2 DNA Laddering Assay
				3.2.3 Annexin V Assay
				3.2.4 LDH Release Assay
		4 Notes
		References
	Chapter 19: Lipoprotein Extraction from Microbial Membrane and Lipoprotein/Lipopeptide Transfection into Mammalian Cells
		1 Introduction
		2 Materials
			2.1 M. salivarium and T. forsythia Cultures
			2.2 Lipoprotein Extraction
			2.3 Lipopeptide Transfection into Macrophages
		3 Methods
			3.1 Bacterial Cultures
				3.1.1 M. salivarium Culture
				3.1.2 T. forsythia Culture
			3.2 Lipoprotein Extraction by TX-114 Phase Separation Method (SeeNote 8)
			3.3 Transfection of the Lipopeptide FSL-1 into the Macrophages
		4 Notes
		References
Part III: Interactions with Other Pathogenic Microorganism and Host Cells
	Chapter 20: Analysis of the Interaction Between HIV and Periodontopathic Bacteria That Reactivates HIV Replication in Latently...
		1 Introduction
		2 Materials
			2.1 Preparation of Culture Supernatant and Bacterial Cells
			2.2 Cell Culture (See Note 1)
			2.3 Stimulation Experiments
			2.4 Immunoblotting Analysis of HIV Antigens
			2.5 Polymerase Chain Reaction (PCR) Analysis of HIV RNA Expression
			2.6 Enzyme-linked Immunosorbent Assay (ELISA) for Detection of the HIV Core Protein p24
			2.7 Analysis of HIV Transcription Activity by Luciferase Assay
		3 Methods
			3.1 Preparation of Culture Supernatant and Bacterial Cells from Periodontal Pathogens
			3.2 Stimulation Experiments (Fig. 1)
			3.3 Viral Replication Assay
				3.3.1 Immunoblotting Analysis of HIV Antigens
				3.3.2 PCR Analysis of HIV RNA Expression
				3.3.3 ELISA for Detection of the HIV Core Protein p24
				3.3.4 Analysis of HIV Transcription Activity by Luciferase Assay
		4 Notes
		References
	Chapter 21: Invasion of Gingival Epithelial Cells by Porphyromonas gingivalis
		1 Introduction
		2 Materials
			2.1 P. gingivalis
			2.2 Gingival Epithelial Cells
			2.3 Confocal Microscopy
			2.4 Cell Staining
		3 Methods
			3.1 Culturing of P. gingivalis
			3.2 Culturing of IHGE Cells
			3.3 Infection of Cells with P. gingivalis
			3.4 Analysis Using Confocal Microscopy
		4 Notes
		References
	Chapter 22: Analysis of Interaction Between Porphyromonas gingivalis and Endothelial Cells In Vitro
		1 Introduction
		2 Materials
			2.1 Bacterial Strains and Growth Conditions
			2.2 Cells and Culture Conditions
			2.3 P. gingivalis Adherence to Endothelial Cells
			2.4 P. gingivalis Invasion into Endothelial Cells
			2.5 P. gingivalis-Induced NO Production
			2.6 P. gingivalis-Induced vWF Release
		3 Methods
			3.1 Bacterial Cell Culture
			3.2 Endothelial Cell Culture
			3.3 Analysis of P. gingivalis Adhesion to Endothelial Cells
			3.4 P. gingivalis Invasion into Endothelial Cells
			3.5 P. gingivalis-Induced NO Production
			3.6 P. gingivalis-Induced vWF Release
		4 Notes
		References
Part IV: Animal Model of Periodontitis
	Chapter 23: Analysis of Experimental Ligature-Induced Periodontitis Model in Mice
		1 Introduction
		2 Materials
			2.1 Animals
			2.2 Ligation
			2.3 Microinjection
			2.4 Bone Analysis
			2.5 Tissue Analysis
			2.6 Molecular Analysis
			2.7 Bacteria Analysis
		3 Methods
			3.1 Breeding
			3.2 Ligation
			3.3 Microinjection
			3.4 Bone Analysis
			3.5 Tissue Analysis
			3.6 Molecular Analysis
			3.7 Bacteria Analysis
		4 Notes
		References
Index