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11 Genomes of Pathogenic Neisseria Species 231

Christoph Schoen, Heike Claus, Ulrich Vogel, and Matthias Frosch

11.1 Introduction 231

11.2 Genomes of Pathogenic Neisseria Species 232

11.2.1 The Flexible Genome Pool 233

11.2.2 Repetitive DNA Sequence Elements Govern Neisserial Biology 236

11.2.2.1 DNA Uptake Sequences, Horizontal Gene Transfer, and Antigenic Diversity 236

11.2.2.2 Simple Sequence Repeats and Phase Variation 237

11.2.2.3 Insertion Sequences and the Regulation of Gene Expression 238

11.2.3 Genome-Wide Mutational Analyses 239

11.2.4 Comparative Genomics 240

11.2.5 Novel Virulence Factors of Meningococci Identified by Genomic Approaches 244

11.3 Future Perspectives 249

12 Genomics of Pathogenic Clostridia and Bacilli 257

Armin Ehrenreich, Gerhard Gottschalk, and Holger Brüggemann

12.1 Genomics of Pathogenic Clostridia spp. 257

12.1.1 Introduction 257

12.1.2 C.perfringens 258

12.1.3 C. tetani 260

12.1.4 C. botulinum 262

12.1.5 C. difficile 263

12.1.6 Conclusions and Perspectives 264

12.2 Genomics of Pathogenic Bacilli 265

12.2.1 Introduction 265

12.2.2 Pathogenic Properties of Bacilli not Belonging to the B. cereus Group 266

12.2.3 Pathogenicity ofB. cereus 266

12.2.4 Pathogenicity ofB. anthracis 268

12.2.4.1 Course of Anthrax 268

12.2.4.2 Virulence Factors of B. anthracis 269

12.2.5 Genome of B. anthracis 270

12.2.5.1 Chromosomal Genes 270

12.2.5.2 Genes Located on Plasmids pXO1 and pXO2 271

12.2.5.3 Regulation of Virulence Genes 272

12.2.5.4 Molecular Diversity in B. anthracis Genomes 272

12.2.5.5 Genome of a Highly Virulent B. cereus Strain Resembling B. anthracis in Pathogenesis 273

12.2.6 Comparison of B. cereus Group Genomes: How Did Pathogenicity Evolve? 273

13 The Genomes of Pathogenic Bartonella Species 281

Carolin Frank, Eva Berglund, and Siv G. E. Andersson 281

13.1 Introduction 281

13.1.1 Bartonella in a Phylogenetic Context 281

13.1.2 Hosts and Vectors for Bartonella Species 282

13.2 Bartonella Species and Pathogenicity 284

13.2.1 Infection of Reservoir and Incidental Host 284

13.2.2 Bartonella Species as Human Pathogens 285

13.3 The Bartonella Genomes 286

13.4 Genomic Islands and Phages 286

13.5 Genomic Islands and Phages in Bartonella Species 287

13.5.1 The B. henselae Prophage 288

13.5.2 B. henselae Genomic Islands and Islets 288

13.5.3 B. quintana Harbors Remnants of the B. henselae Islands 290

13.5.4 Role of Phages and Islands in the Evolution of Bartonella 290

13.6 The Chromosome II-Like Segment in Bartonella 291

13.6.1 Type IV Secretion Systems in Bartonella Species 292

13.6.2 The virB-D4 Operon 293

13.6.3 The trw Operon 293

13.7 B. quintana's Evolution into a Human Pathogen 294

13.8 Conclusions and Future Perspectives 295

14 Pathogenomics of Gastric and Enterohepatic Helicobacter Species 301

Sebastian Suerbaum, Sandra Schwarz, and ChristineJosenhans

14.1 Introduction 301

14.2 Helicobacter pylori 302

14.2.1 Key Features of the H. pylori Genome Related to Pathogenesis 302

14.2.1.1 Colonization Factors: Urease and Motility 302

14.2.1.2 Phase Variation 302

14.2.1.3 The H. pylori Outer Membrane Protein Family 303

14.2.1.4 Intraspecies Variation of H. pylori Genomes 303

14.2.1.5 The cag Pathogenicity Island 304

14.2.1.6 Nucleotide Sequence Variation in H. pylori 305

14.3 Helicobacter hepaticus 305

14.3.1 The HHGI1 Genomic Island 306

14.3.2 Other Putative H. hepaticus Virulence Factors 307

14.4 Genome Comparisons of Gastric and Enterohepatic Helicobacter Species with Related Bacteria 307

14.5 Outlook 308

15 Genomics of the Opportunistic Pathogen Legionella pneumophila 315

Christel Cazalet and Carmen Buchrieser

15.1 The Genus Legionella: Epidemiology, Life Cycle, and Pathogenesis 315

15.2 Genomics of Legionella pneumophila 316

15.3 Specific Features of the Legionella Genomes 318

15.3.1 Eukaryotic-like Proteins in Legionella pneumophila: Modulation of Host Functions? 318

15.3.2 Secretion Machineries of L. pneumophila: Central to Its Life and to Pathogenesis 324

15.3.2.1 Type IV Secretion Systems in Legionella 325

15.3.2.1.1 The dot/icm Type IVB Secretion System 325

15.3.2.1.2 The lvh Type IVA Secretion System 326

15.3.2.2 A Putative Type I Secretion System in Legionella 326

15.3.2.3 A Type II Secretion System in Legionella 327

15.3.2.4 Secretion Across the Cytoplasmic Membrane 327

15.3.2.5 A Putative Type V Secretion System (Autotransporter) Specific to Strain Paris 328

15.3.3 Comparative Genomics: Diversity of the Species L. pneumophila 329

15.3.3.1 Genomic (Pathogenicity) Islands in the L. pneumophila Genomes 329

15.3.3.2 Plasmids and Genetic Diversity of L. pneumophila 331

15.4 Conclusions 333

16 Genomics of Listeria monocytogenes 339

Michael Kuhn and Werner Goebel

16.1 Introduction: From Pregenomics to Postgenomics 339

16.2 Listeria monocytogenes: A Facultative Intracellular Pathogen 339

16.3 Listeria monocytogenes Genetics in the Pregenomic Era: Identification and Characterization of Important Virulence Factors 340

16.3.1 Internalins and the Invasion of Nonprofessional Phagocytic Cells 340

16.3.2 Listeriolysin O and Two Listerial Phospholipases Allow Escape from the Phagocytic Vacuole 342

16.3.3 Intracellular Motility and Cell-to-Cell Spread: The Surface Protein ActA 343

16.3.4 PrfA and the Regulation of Virulence Gene Expression 344

16.4 Genome Sequence of L. monocytogenes and Its Comparison with the Closely Related L. innocua 346

16.5 Genomic Approaches to Studying the other Members of the Genus Listeria 348

16.6 Evolutionary Aspects 349

16.7 Identification of Listerial Virulence Factors in the Postgenomic Era 351

16.7.1 Internalins and Other Surface Proteins 351

16.7.2 Growth in the Host Cell Cytoplasm 352

16.7.3 Resistance to Bile 353

16.7.4 Two-component Systems and the Regulation of Virulence Gene Expression 354

16.7.5 Vitamin B12 Biosynthesis and Anaerobic Use of Ethanolamine 354

16.8 Proteomics 355

16.9 Transcriptomics 356

16.10 Conclusions 358

III Genomics of Pathogens and Their Hosts: Applications 367

17 Genomics of Viruses 369

Esteban Domingo, Alejandro Brun, José Ignacio Nunez, Juan Cristina, Carlos Briones, and Cristina Escarmis

17.1 Introduction: Wide Scope of Virogenomics 369

17.2 Retrieving Information 371

17.3 Applications of Data Banks to Virology 374

17.4 Beyond Reference Strains: Towards a Second-Generation Virogenomics? 379

17.5 Virogenomics Through Microarrays 382

18 Genomics of Pathogenic Fungi 389

Gerwald A. Kohler, Alan Kuo, George Newport, and Nina Agabian

18.1 Introduction 389

18.2 Genomics of Primary Fungal Pathogens 390

18.2.1 Histoplasma 390

18.2.2 Coccidioides 396

18.2.3 Blastomyces and Paracoccidioides 396

18.3 Genomics of Opportunistic Fungal Pathogens 397

18.3.1 Aspergillus 397

18.3.2 Cryptococcus 398

18.3.3 Pneumocystis 401

18.3.4 Microsporidia 402

18.3.5 Candida 403

18.4 The Tool Box for Functional Genomics 406

18.4.1 Expression Analysis 407

18.4.2 Transformation and Mutagenesis 407

18.5 Fungal Virulence - From the Genomic Point of View 410

18.6 Conclusion 411

19 Genomics of Pathogenic Parasites 417

Gabriele Pradel and Thomas James Templeton

19.1 Exploring the Genomes of Pathogenic Protozoans 418

19.2 The Shaping of the Proteomes of the Pathogenic Protists 420

19.3 Role of Horizontal Gene Transfer in Protozoan Genome Plasticity 421

19.4 The Apicomplexa 422

19.4.1 Plasmodium, the Malaria Parasite 423

19.4.2 Cryptosporidium 427

19.4.3 Toxoplasma 428

19.5 The Pathogenic Kinetoplastids 429

19.5.1 Trypanosoma 430

19.5.2 Leishmania 431

19.6 The Pathogenic Diplomanad Giardia and the Parabasalid Trichomonas 431

19.7 Postgenomic Strategies and the Search for Cure 433

19.7.1 Gene Expression Analysis 434

19.7.2 Proteomics 434

19.7.3 Drug and Vaccine Development 435

19.7.4 Vector Genetics 436

20 Model Host Systems: Tools for Comprehensive Analysis of Host-Pathogen Interactions 445

Michael Steinert and Gernot Glöckner

20.1 Introduction 445

20.2 Host-Pathogen Interactions 446

20.3 Arabidopsis thaliana: A Plant as a Model for Human Disease 447

20.4 Dictyostelium discoideum: Perspectives from a Social Amoeba 448

20.5 Caenorhabditis elegans: Answers from a Worm 449

20.6 Drosophila melanogasten A Fruitful Model 450

20.7 Danio rerio: Fishing for Knowledge 451

20.8 Mus musculus: Of Mice and Men 451

20.9 Clean Models and Dirty Reality 453

21 Expression Analysis of Human Genes During Infection 457

Erwin Bohn and Ingo B. Autenrieth

21.1 Introduction 457

21.2 Comparison of Gene Expression Profiles of Macrophages and Dendritic Cells In Vitro Upon Infection with Different Pathogens 458

21.3 Septicemia 460

21.4 Gene Expression in Epithelial Cells Modulated by Bacteria 461 21.4.1 Helicobacter pylori 462

21.4.2 Yersinia enterocolitica 464

21.4.3 Pseudomonas aeruginosa 466

21.4.4 Bartonella henselae 468

21.5 Common Signatures 469

21.6 Genetic Polymorphisms and Mutations Affect Gene Expression: Impact on Infection Susceptibility and Infection Course 471

21.7 Concluding Remarks 473

22 Pathogenomics: Application and New Diagnostic Tools 481

Sören Schubert and Jürgen Heesemann

22.1 Introduction: "In Our Hands" 481

22.2 Microbiological Diagnostics of Bacterial Pathogens: Aims, Tasks, and Current Limitations 482

22.3 The Pregenomic Era: Conventional and Molecular Methods in Microbiological Diagnostics 483

22.3.1 Conventional Culture-Based Methods in Microbiological Diagnostics 483

22.3.2 Molecular Microbiological Diagnostic Methods 484

22.3.2.1 Typing of Bacterial Isolates Using 16S-rRNA 484

22.3.2.2 Fluorescence In Situ Hybridization 484

22.3.2.3 PCR Methods for Microbial Diagnostics 485

22.4 The Postgenomic Era: Use of DNA Microarrays in the Diagnosis of Infectious Diseases in Humans and Animals 487

22.4.1 DNA Arrays: Platforms, Techniques and Targets 487

22.4.2 Detection and Typing of Microbial Pathogens 488

22.4.3 Pathoarrays 489

22.4.4 16S-/23S-rDNA Arrays 494

22.4.5 Detection of Antibiotic Resistance in Microbial Pathogens Using Microarray Technology 494

22.5 Microarray Technology in Bacteria: Further Areas of Applications 495

22.5.1 Gene Expression Microarrays and Host-Pathogen Interaction 495

22.5.2 DNA Microarray Technology in Food Technology 496

22.5.3 DNA Microarray Technology in Environmental Microbiology 496

22.5.4 Pathogenomic Tools (Microarrays) in the Diagnosis of Microbiologic Agents as Bioweapons 497

22.6 Current Limitations on the Use of DNA Microarrays in Diagnostics in Medical Microbiological Laboratories 498

22.7 Final Remarks 498

23 The Search for New Antibiotics 505

Harald Labischinski, Christoph Freiberg, and Heike Brotz-Oesterhelt

23.1 The Need for Novel Antibiotics 505

23.2 Where Will the New Antibiotics Come From? 507

23.2.1 The Past 507

23.2.2 The Present 509

23.2.3 Future Directions 511

23.3 Contributions ofGenomic Technologies to Antibacterial Research 513

23.3.1 Target Identification and Validation 513

23.3.2 Target Prioritization 517

23.3.3 Genetic Tools for Drug Screening and Mode-of-Action Determination 517

23.3.4 Genome-Wide Expression Profiling for Mode-of-Action Characterization 520

23.3.5 Outlook for Genomic Technologies for Antibiotic Drug Discovery 521

23.4 Alternative Approaches in Antibacterial Drug Discovery 521

23.4.1 Targeting the Resistance Mechanism 522

23.4.2 Extremely Narrow-Spectrum Drugs 523

23.4.3 Phage Therapies and other Bacteriolytic Approaches 524

23.4.4 Strategies for Reducing Virulence and/or Influencing Pathogenesis 525

24 Reverse Vaccinology: Revolutionizing the Approach to Vaccine Design 533

Laura Serino, Mariagrazia Pizza, and Rino Rappuoli 533

24.1 Impact of Genomics on Vaccine Design 533

24.2 MenB Vaccine Approach by Reverse Vaccinology 535

24.3 Following the MenB Experience: Other Pathogens 538

24.4 Functional Genomics 539

24.5 Gene Expression In Vivo: IVETand STM 540

24.6 Transcriptome Analysis and Comparative Genomics 541

24.7 Proteomics and Vaccine Design 546

24.8 Conclusions 546

Index 555

Preface

In the year 1995, the first full genome sequence of a free-living-organism, the bacterium Haemophilus influenzae strain Rd, was published. This publication, which appeared in the journal Science, represented the starting point for a new field in molecular biology called genomics. Today, 10 years later, complete genome sequences of almost all the major pathogenic microbes have been determined. As a consequence, a new discipline has arisen, which has been named "pathoge-nomics." As the name implies, pathogenomics is the analysis at the genomic level of the processes involved in bacterial pathogenesis caused by the interaction of pathogenic microbes and their hosts.

The present volume is the first handbook to be entirely devoted to the newly established discipline of pathogenomics. We are very grateful to our colleagues for their input, especially those associated with the German Pathogenomics competence network, established by the German Federal Ministry of Science and Education to analyze pathogenic microbes at the genomic level. Werner Goebel in particular - the network's speaker, who contributed the Foreword to this book's preface - has influenced the entire discipline with his spirit and his vision.

Our thanks are due to the staff at Wiley-VCH, most notably Andrea Pillmann, who encouraged us to put this book together. We are also very grateful to all our authors for their contributions to this important project.

Würzburg, September 2005

Ulrich Dobrindt, Jörg Hacker

List of Contributors

Nina Agabian

University of California San Francisco Department of Cell and Tissue Biology 521 Parnassus, Box 0640 San Francisco, CA 94143-0640 USA

Siv Andersson

Uppsala University

Evolutionary Biology Center

Department of Molecular Evolution

Norbyvägen 18C

752 36 Uppsala University

Sweden

Helene Andrews-Polymenis

Department of Medical Microbiology and Immunology

College of Medicine

Texas A&M University HSC

407 Reynolds Medical Building

College Station, TX 77843-1114

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