Bacterial Pathogenesis: Methods and Protocols

Cover......Page 1
FrontMatter......Page 2
BookSeries......Page 3
TitlePage......Page 4
CopyRight......Page 5
Preface......Page 6
Contents......Page 8
Contibutors......Page 10
List of Color Plates......Page 14
Part I: MECHANISMS OF BACTERIAL PATHOGENESIS......Page 17
Introduction......Page 19
Phenol--Water Technique ch01:bib16......Page 21
Proteinase K Digested, Phenol--Water Extracted LPS......Page 22
Small-Scale Preparations of LPS or LOS......Page 23
LOS and LPS Acrylamide Gel Electrophoresis......Page 24
Silver Staining Technique for LPS and LOS Gels ch01:bib23......Page 25
Isolation of Lipid A ch01:bib24......Page 26
Notes......Page 27
Introduction......Page 31
Second-Dimension Gel Electrophoresis......Page 33
Isolation of CSPs......Page 34
Isoelectric Focusing......Page 35
Second-Dimension Gel Electrophoresis......Page 36
Protein Detection, Quantitation, and Analysis......Page 37
Notes......Page 38
Introduction......Page 41
Cell Culture, Cell Lysis, and Protein Preparation......Page 43
Difference Gel Electrophoresis Labeling of Proteins......Page 44
Protein Detection......Page 45
Methods......Page 46
Protein Preparation......Page 50
 CyDye DIGE Minimal Labeling (for Extracellular Proteins)......Page 51
2D Gel Electrophoresis......Page 52
Protein Detection......Page 54
Protein Identification by Mass Spectrometry (see Fig. 1)......Page 55
Quantitation and Bioinformatic Approaches......Page 56
Notes......Page 59
Introduction......Page 62
Labeling of DNA......Page 63
Genomic DNA Isolation and Digestion......Page 64
Labeling of DNA (Prime-A-Gene Labeling System; Promega Modified Protocol)......Page 65
Scanning of Slides and Data Analysis......Page 66
Notes......Page 67
Introduction......Page 71
Detection and Inhibition of N-Acylated Homoserine Lactone Quorum Sensing......Page 73
AHL Signal, Inducer, or Antagonist Extraction and Preparation......Page 74
Inhibition of AHL Quorum-Sensing Phenotypes......Page 76
Detection of AI-2 Activity or Inhibition......Page 78
Inhibition of Hemolysin Assay with Signal Antagonist......Page 80
Notes......Page 81
Introduction......Page 85
Nucleic Acids......Page 86
Nucleic Acid Cloning Materials......Page 87
Construction of Genetic Manipulation Plasmids......Page 88
Electro-Transformation (see Note 10)......Page 91
Notes......Page 92
Introduction......Page 101
Competent Cell Preparation......Page 104
Methods......Page 105
Identification of Transposon Insertion Site......Page 106
Notes......Page 107
Introduction......Page 113
Materials......Page 114
Purification of PIA......Page 115
PIA Detection by Immunological Assays......Page 117
Analysis and Detection of PIA by Chromatography......Page 118
Notes......Page 119
Part II: HOST-PATHOGEN INTERACTION......Page 123
Introduction......Page 125
Isolation of Human PMNs and Serum, and Phagocytosis Assays......Page 126
cDNA Synthesis of S. aureus RNA......Page 127
Hybridization of S. aureus cDNA to Affymetrix GeneChips......Page 128
GeneChip Scanning and Conversion of Image Files......Page 129
Phagocytosis of S. aureus by Human PMNs......Page 130
Purification of RNA from S. aureus During/After Phagocytosis by Human PMNs......Page 131
DNase Digestion and Clean-Up of S. aureus RNA......Page 132
Labeling Fragmented cDNA with Biotin--ddUTP......Page 133
Processing Affymetrix GeneChips......Page 134
Scanning......Page 135
Notes......Page 136
Introduction......Page 139
Materials......Page 140
Preparation of Standard Curve......Page 142
Preparation of Unknown Samples......Page 143
Data Analysis......Page 144
Notes......Page 145
Introduction......Page 149
Culture and Infection of Primary Macrophages......Page 150
Immunofluorescence Microscopy......Page 151
Methods......Page 152
Culture of Bone Marrow-Derived Macrophages......Page 154
Macrophage Infections......Page 156
Immunofluorescence Microscopy......Page 157
Phagosomal Integrity Assay ch11:bib12......Page 158
Notes......Page 159
Introduction......Page 163
Microscopy Supplies......Page 164
Preparation of Acid-Washed Coverslips......Page 165
Cultivation of Helicobacter pylori......Page 166
Synchronized Phagocytosis......Page 167
Differential Staining of Bound and Ingested Bacteria......Page 168
Mounting Stained Coverslips onto Slides......Page 169
Microscopy and Data Analysis......Page 170
Notes......Page 171
Introduction......Page 175
Anaplasma phagocytophilum Culture and Propagation in HL60 Cells......Page 176
Incubation of Bacteria with Neutrophils......Page 177
Anaplasma phagocytophilum Culture and Propagation in HL60 Cells......Page 178
Isolation of Cell-Free Anaplasma phagocytophilum......Page 180
Bacteria Labeling and Modifications......Page 181
Incubation of Bacteria with Neutrophils......Page 182
Notes......Page 183
Introduction......Page 189
Conventional TEM......Page 191
Immune Labeling with Quantum Dots......Page 193
Conventional TEM......Page 194
Conventional TEM......Page 195
Conventional SEM......Page 197
Immune Labeling with Quantum Dots......Page 198
Notes......Page 201
Introduction......Page 205
Coxiella burnetii Propagation and Purification......Page 206
Quantitative PCR Coxiella burnetii Growth Assay......Page 207
Methods......Page 208
Infection of Vero Cells with Coxiella burnetii......Page 209
PBMC Isolation and Culture of Monocyte-Derived Macrophages......Page 210
Enumeration of Coxiella burnetii Replication......Page 211
Notes......Page 213
Introduction......Page 217
Materials......Page 219
Invasion......Page 221
Immunofluorescence ``Inside-Outside\'\' Assay......Page 222
Notes......Page 223
Introduction......Page 229
Materials......Page 231
Identification of DNA Damage......Page 232
Identification of Lipid Damage......Page 233
Identification of Lipid Peroxidation Intermediates......Page 235
Part III: ANIMAL MODEL OF BACTERIAL INFECTION......Page 239
Jagath L. Kadurugamuwa and Kevin P. Francis......Page 241
Introduction......Page 242
Generation of Bioluminescent Bacteria......Page 243
Transformation Procedure for Gram-positive Bacteria (Staphylococcus aureus Methodology)......Page 244
In vitro Catheter-Associated Biofilm......Page 245
Experimental Model of Infection......Page 246
Catheter-Associated Urinary Tract Infections......Page 248
Central Venous Catheter-Associated Infection in Mice......Page 250
Experimental Model of Infection......Page 251
Notes......Page 252
Introduction......Page 257
Instillation......Page 258
Recovery......Page 259
Instillation......Page 260
Treatment......Page 261
Notes......Page 264
Introduction......Page 271
Blood Draws......Page 273
Preparation of Bacterial Inoculum......Page 274
Cynomolgus Macaque Screens for Group Selection......Page 275
Sample Isolations and Clinical Scoring of Disease......Page 277
Post-experiment Antibiotic Treatment of Animals......Page 280
Notes......Page 281
Part IV: IDENTIFICATION OF THERAPEUTIC TARGETS......Page 285
Introduction......Page 287
Methods of Target Identification......Page 288
Oldies but Goodies......Page 289
Translation......Page 290
Other Validated Antibacterial Targets......Page 291
A Selection of Novel Targets---Innovative Approaches......Page 292
Virulence as a Target -- a Kinder and Gentler Form of Disarmament......Page 293
Challenges for Target-Driven Antibacterial Drug Discovery -- How to Hit the Bullseye......Page 294
Concluding Thoughts......Page 296
Introduction......Page 301
Variable Number Tandem Repeats......Page 302
DNA Isolation......Page 307
PCR......Page 308
Analysis/Interpretation......Page 310
Other Genotyping Techniques......Page 313
Discussion......Page 315
index......Page 323
ColorInsert......Page 328