WILEY-VCH Verlag GmbH & Co. KGaA

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Universität Würzburg

Institut für Molekulare Infektionsbiologie

Röntgenring 11

97070 Würzburg


Dr. Ulrich Dobrindt

Universität Würzburg

Institut für Molekulare Infektionsbiologie

Röntgenring 11

97070 Würzburg


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The determination of the genome sequences of many prokaryotic and eukaryotic organisms, together with the high-throughput techniques (transcriptomics, pro-teomics, metabolomics, interactomics, etc.) and the powerful tools of bioinfor-matics, have opened up to us all new perspectives for the deeper understanding of the basic mechanisms of life.

Because of the small size of bacterial genomes and the collinearity of their genetic information, prokaryotes are particularly suited to genome-based in-depth analysis of the essential processes which allow these microorganisms to survive and replicate in many different environments. Some of them can even multiply in those parts of the human body which are normally well protected by highly developed antimicrobial defense mechanisms. This latter property is the most outstanding evolutionary achievement of microbial pathogens that are capable of causing infectious diseases in humans.

The present monograph summarizes the state of genome-based research on some of the most important bacterial (and to a lesser extent viral and fungal) human pathogens today. The term pathogenomics has been coined for this new branch of microbiology. Not surprisingly, the major focus of pathogenomics was first on those human bacterial pathogens that (a) can cause major epidemics, especially in developing countries (e.g., Shigella spp. or Vibrio cholerae); (b) represent major health problems in almost all human societies in the form of food contaminants and/or agents of nosocomial infections (e.g., pathogenic Enterobacteria-ceae, staphylococci, and streptococci); and (c), due to their frequent occurrence in humans (e.g., Helicobacter pylori and Mycobacterium tuberculosis) represent life-threatening problems not only for many people in developing countries, but also for immune-compromised and elderly persons in all the industrialized countries. These microorganisms therefore also represent the major objects described in this book.

A primary goal of pathogenomics is to use the new experimental tools in combination to unravel those genes - and gene arrangements - of pathogens that are essential for causing disease, and thereby to shed new light on the evolution, interbacterial gene transfer, and distribution of these virulence genes among bacterial populations. This area of pathogenomics, which is already highly advanced, makes up the largest part of this monograph.

The other, certainly not less important goal is to study the functional significance of the pathogen-specific genes in the infection process. In the years to come, the enormous amount of genetic information that has piled up in the last 10 years from the sequencing of the genomes of most of the important bacterial pathogens (and their closely related nonpathogenic environmental counterparts) will need to be functionally analyzed. It is anticipated that this functional pathoge-nomics will finally unravel the mechanisms of differential and coordinated regulation of the virulence genes, the structural, molecular, and physiological functions of their gene products, which will lead to a more comprehensive view on the pathogenic microorganisms. Some aspects of this most interesting future line of microbial research that can be expected to become the mainstream in future pathogenomics are already addressed in some of the chapters of this book.

Infections are the outcome of the encounter between the microbial pathogen and its host. Describing microbial infections in molecular terms therefore requires among other things a profound understanding of the host cell responses. The availability today of the genome sequences of man and some of the major model hosts in which microbial infections can be experimentally studied (the mouse in particular, but also alternative hosts such as amebae, the nematode Cae-norhabditis elegans, Drosophila melanogaster, or the slime mold Dictyostelium) together with the new genetic and bioinformatics tools open up new avenues towards uncovering at least some of the basic host cell responses.

Whilst cellular microbiology has already delivered an enormous set of valuable host-response information at the cellular level, the new in vivo imaging techniques, siRNA technology, and the routine genetic manipulations of some of the abovementioned model hosts may now allow such molecular infection studies to be performed in real hosts. This monograph devotes several chapters to this important aspect of pathogenomics.

Science and, especially, the public and political worlds expect pathogenomics to provide novel ideas and strategies to combat infectious diseases through the rational design of better diagnostic tools and novel anti-infectives and vaccines. Indeed, some promising new developments deriving from pathogenomics are already visible and are in part outlined in some chapters ofthis monograph. These new approaches will most undoubtedly help in fighting the most dangerous infectious agents which still claim the highest death toll in mankind. However, scientists should be modest in their promises relating to the possibilities that arise from this exciting new field of science, since one major lesson which the attentive and critical reader of this monograph will quickly learn is the enormous genetic flexibility and adaptation potential of pathogenic microorganisms - and that means they will keep us busy for decades to come despite all the obvious successes of pathogenomics.

The book thus offers a representative view of the present state of the art in the new research area of pathogenomics. It is a valuable and reasonably comprehensive source of information for scientists and advanced students who wish to become acquainted with this most exciting field of modern microbiology.

Würzburg, October 2005

Werner Goebel


Foreword V

Preface XIX

List of Contributors XXI

Color Plates XXVII

I Methods 1

1 Bioinformatics: Data Mining Among Genome Sequences 3

Susanne Kneitz and Thomas Dandekar

1.1 Systematic Genome Analysis of Pathogens as a Basis for Pharmacogenomic Strategies 3

1.2 Direct Sequence Annotation Tools for Functional Genomics 4

1.3 Identification of Protein Function 4

1.4 Obtaining Protein Information from a Domain Server 5

1.5 Pathway Analysis 6

1.6 Network Analysis 7

1.7 Adaptation in Time and to Stimuli 8

1.7.1 Experimental Design for Microarray Analysis 8

1.7.2 Data Analysis 9

1.8 Pathogen-Specific Challenges 13

1.9 Pathogen Adaptation Potential 14

1.10 The Fight Against Resistance 14

1.11 Drug Design and Antibiotics 15

1.12 Annotation Platforms Suitable for Pathogenomics 15

1.13 Conclusions 17

2 Transcriptome Analysis: Towards a Comprehensive Understanding of Global Transcription Activity 21

Ben Sidders and Neil Stoker

2.1 Introduction 21

2.2 Development of Transcriptomics 21

2.2.1 From Genomics to Functional Genomics 21

2.2.2 From Gene to Whole Genome 22

2.3 Introducing the Microarray 23

2.3.1 What Is a Microarray? 23

2.3.2 The Affymetrix Gene Chip 23

2.3.3 The Spotted Microarray 24

2.4 Microarray Methods 25

2.4.1 Experimental Design 25 Type of Experiment 25 Replicates 28

2.4.2 RNA Extraction 28

2.4.3 Labeling/Reverse Transcription 30

2.4.4 Hybridization 31

2.4.5 Scanning 31

2.5 Data Normalization and Analysis 32

2.5.1 Image Quantification 32

2.5.2 Data Processing 33

2.5.3 Data Analysis 34 Detection of Differential Expression 34 Pattern Recognition 35 Graphical Representations 35

2.5.6 Microarray Analysis Tools 36

2.5.7 Microarray Follow-Up 36

2.5.8 Data Storage and Reanalysis 37

2.6 Transcriptomics: Where We Are Now and What's to Come 37

3 Physiological Proteomics of Bacillus subtilis and Staphylococcus aureus: Towards a Comprehensive Understanding of Cell Physiology and Pathogenicity 43

Michael Hecker and Susanne Engelmann

3.1 Introduction 43

3.2 Proteomics of Bacillus subtilis: The Gram-positive Model Organism 46

3.2.1 The Vegetative Proteome 46

3.2.2 Proteomes of Nongrowing Cells: Proteomic Signatures of Stress/Starvation Stimuli 47

3.3 Physiological Proteomics of Staphylococcus aureus 53

3.3.1 The Postgenome Era of S. aureus 53

3.3.2 Proteomes of Growing and Nongrowing Cells 56

3.3.3 Extracellular Proteins and Pathogenicity Networks 62 3.4 Outlook: Second Generation Proteomics and

New Fields in S. aureus Physiology and Infection Biology 65

4 Impact of Genome Sequences on Mutational Analysis of Fungal and Bacterial Pathogens 69

Vladimir Pelicic and Xavier Nassif 69

4.1 The Long Road from Sequence to Function 69

4.2 Classical Genetics Still at the Forefront in the Postgenome Era 70

4.2.1 Reverse Genetics 70

4.2.2 Transposon Mutagenesis 71

4.3 Genome-Scale Mutational Analyses 72

4.3.1 Saccharomyces cerevisiae 73

4.3.2 Bacterial Workhorses: E. coli and Bacillus subtilis 75

4.3.3 Bacterial Pathogens 75 Mycoplasma Species 76 Pseudomonas aeruginosa 76 Staphylococcus aureus 77 Neisseria meningitidis 77

4.4 Conclusion 79

II Genomics of Pathogenic Bacteria 83

5 Pathogenomics of Escherichia coli and Shigella Species 85

Ulrich Dobrindt and Jörg Hacker

5.1 Introduction 85

5.2 Comparative Genomics of Shigella 86

5.3 Comparative Genomics of Escherichia coli 92

5.3.1 Comparison of Complete Genome Sequences 92

5.3.2 Comparative Genomics Using DNA Arrays 94

5.3.3 Mobile Genetic Elements and Evolution of Pathogenic E. coli 95

5.3.4 Genomic Islands/Pathogenicity Islands 95

5.3.5 Plasmids and Bacteriophages 99

5.3.6 Genetic Diversity Among Extraintestinal Pathogenic E. coli 100

5.4 Conclusions 101

6 Pathogenomics of Salmonella Species 109

Helene Andrews-Polymenis and Andreas J. Bäumler

6.1 Introduction 109

6.2 Salmonella Signature Genes 109

6.3 Subspecies I Signature Genes 112

6.4 Host Restriction 115

7 Pathogenomics of Enterococcus faecalis 125

Janet M. Manson and Michael S. Gilmore

7.1 Introduction 125

7.2 Enterococcal Pathogenesis 125

7.3 Genome Sequence of E. faecalis 126

7.3.1 Mobile Elements, Acquired DNA, and Antimicrobial Resistance 127

7.3.2 Environmental Adaptation and Stress Response 131

7.3.3 Survival In Vivo 133

7.3.4 Potential Virulence Factors 134 Hemolysins, Proteases, and other Enzymes 134 Cell-Wall-Associated Virulence Factors 136

7.3.5 Pathogenicity Island of E. faecalis 138

7.4 Conclusions and Future Perspectives 140

8 Genomics of Streptococci 149

Joseph J. Ferretti and W. Michael McShan

8.1 Introduction 149

8.2 Bacterial Genomes 152

8.2.1 Pyogenic Group 152 Streptococcus pyogenes 152 Virulence Factors 153 Horizontal Gene Transfer 154 Streptococcus agalactiae 155 Group C (GCS) and Group G Streptococci 156 Streptococcus uberis 157

8.2.2 Bovis Group 157 Streptococcus bovis and Streptococcus suis 157

8.2.3 Mitis Group 158 Streptococcus pneumoniae 158 Streptococcus mitis, Streptococcus sanguis, and Streptococcus gordonii 159

8.2.4 Anginosus and Salivarius Group 159 Streptococcus salivarius 159 Streptococcus thermophilus 160

8.2.5 Mutans Group 160 Streptococcus mutans and Streptococcus sobrinus 160

8.2.6 Other Organisms: Enterococcus faecalis

8.2.7 Comparative Genomics 161 8.3 Streptococcal Genomic Bacteriophages

8.3.1 Prophages and Streptococcal Genomes

8.3.2 GAS Genome Prophages 163 Prophages and Virulence Factors 163 Prophage Attachment Sites and Host Biology 165 Prophage Diversity 166

8.3.3 Prophages Associated with other Streptococcal Species 166

9 Pathogenic Staphylococci: Lessons from Comparative Genomics 175

Knut Ohlsen, Martin Eckart, Christian Hüttinger, and Wilma Ziebuhr

9.1 Introduction 175

9.2 Comparative Genomics of S. aureus 176

9.2.1 Overall Genome Structure 177

9.2.2 Core Genome 178 Metabolism 178 Information Pathways 180 Virulence Factors 181

9.2.3 Accessory Genome 184 Pathogenicity Islands 185 Staphylococcal Cassette Chromosome 190 Bacteriophages 192 Plasmids 194

9.3 Staphylococcus epidermidis 195

9.3.1 Genomic Islands 197

9.3.2 Phage SPb and other Bacillus Genes 197

9.3.3 Virulence Factors 197

9.3.4 Staphylococcal Cassette Chromosome 198

9.3.5 Adherence and Biofilm Formation 199

9.3.6 Insertion Sequences 201

9.4 Concluding Remarks 202

10 Pathogenomics: Insights into Tuberculosis and Related Mycobacterial Diseases 211

Alexander S. Pym, Stephen V. Gordon, and Roland Brosch

10.1 Introduction 211

10.2 Molecular Basis of Pathogenicity 212

10.3 Evolution ofthe M. tuberculosis Complex 216

10.4 Some Metabolic Insight from the Genome Sequences 220

10.5 Other Major Mycobacterial Human Pathogens 222

10.5.1 Mycobacterium leprae 222

10.5.2 Mycobacterium ulcerans 223

10.6 Concluding Remarks 224

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