BSL3 and BSL4 Agents: Proteomics, Glycomics and Antigenicity

Edited by leading experts in the EU and the US, this book provides a unique coverage of novel technology approaches for the detection of highly related variants of dangerous agents and novel therapy options. A must–have for all professionals dealing with BSL3 and/or BSL 4 agents

Spis treści:
Preface XIII
List of Contributors XV
1 Introduction: Application of Proteomic Technologies for the Analysis of Microbial Infections 1
Jiri Stulik and Patrick Butaye
1.1 Introduction 1
1.2 Search for New Factors of Virulence and Potential Diagnostic Markers 2
1.3 Search for New Vaccine Candidates 3
1.4 Analysis of Post–Translational Modifications of Bacterial Proteins and Protein–Protein Interactions 3
1.5 Conclusions 5
References 5
Part One Basic Proteomic Methods 7
2 Separation of Proteins and Peptides 9
Ludovit Skultety
2.1 Introduction 9
Acknowledgment 24
References 24
3 Basic Mass Spectrometric Approaches 29
Lenka Hernychova and Martin Hubalek
3.1 Introduction 29
3.2 Ionization 30
3.3 Mass Analyzers 32
3.4 Protein Identifi cation 37
3.5 Conclusion 39
Acknowledgments 40
References 40
4 Quantitative Mass Spectrometric Approaches 43
Juraj Lenco and Vojteˇch Tambor
4.1 Introduction 43
4.2 iTRAQ Analysis of Bacterial Pathogens 47
References 52
5 BN–PAGE of Microbial Protein Complexes 55
Jiri Dresler and Jana Klimentova
5.1 Introduction 55
5.2 Methods for Studying Protein–Protein Interactions 55
5.3 Blue Native Polyacrylamide Gel Electophoresis 56
5.4 Evaluation of BN–PAGE – Staining, MS, Western Blotting 60
5.5 BN/SDS–PAGE of ATP S ynthase of Francisella tularensis 62
5.6 Conclusion 63
Acknowledgment 63
References 64
6 Analysis of Francisella tularensis Glycoproteins 67
Lucie Balonova and Lenka Hernychova
6.1 Introduction to Post–Translational Modifi cations in Prokaryotes 67
6.2 Methodology 68
6.3 Bioinformatics 70
6.4 Application of Glycoproteomic Approach Utilizing ProQ–Emerald and DIG Glycan Kits to Francisella tularensis (F. tularensis) 70
6.5 Results 73
6.6 Conclusion 74
Acknowledgments 76
References 76
Part Two Identification of Proteins and Glycans from Microorganisms as Candidate Molecules for Use in Detection/Diagnosis, Therapy, and Prophylaxis 79
7 Comparative Proteome Analysis of Strains with Differential Virulence 81
Martin Hubalek and Ivona Pávková
7.1 Introduction 81
7.2 Methods 82
7.3 Results 84
7.4 Discussion 86
References 91
8 Analysis of Francisella tularensis Acetonitrile Extracts 95
Lenka Hernychova, Martin Hubalek, and Jana Udrzalova
8.1 Introduction 95
8.2 Material and Methods 96
8.3 Results 98
8.4 Conclusions 102
Acknowledgments 103
References 103
9 Analysis of Culture Filtrate Proteins of Francisella tularensis 107
Klara Konecna, Martin Hubalek, and Lenka Hernychova
9.1 Introduction 107
9.2 Materials and Methods 108
9.3 Results 109
9.4 Discussion 112
Acknowledgments 113
References 113
10 Lipopolysaccharides of Coxiella burnetii: Chemical Composition and Structure, and Their Role in Diagnosis of Q Fever 115
Rudolf Toman and Pavol Vadovicˇ
10.1 Introduction 115
10.2 Lipopolysaccharides of C. burne tii 116
10.3 Conclusion 121
Acknowledgments 121
References 121
11 Mimivirus Possesses Anonymous and Unique Gene Products Endowed for Antigenic Properties 125
Patricia Renesto and Didier Raoult
11.1 Introduction 125
11.2 Material and Methods 126
11.3 Results 127
References 128
12 Detection of Differentially Modifi ed Pathogen Proteins by Western Blot after 2D Gel Electrophoresis and Identifi cation by MALDI–TOF/TOF 131
Fred Fack, Julia Kessler, Patrick Pirrotte, Jacques Kremer, Dominique Revets, Wim Ammerlaan, and Claude P. Muller
12.1 Introduction 131
12.2 Materials and Methods 132
12.3 Results 134
12.4 Discussion 137
References 138
13 Composition and Structure of Lipid A of the Intracellular Bacteria Piscirickettsia salmonis and Coxiella burnetii 139
Pavol Vadovicˇ, Robert Ihnatko, and Rudolf Toman
13.1 Introduction 139
13.2 Composition and Structure of Lipid A of P. salmonis 140
13.3 Composition and Structure of Lipid A of C. burnetii 141
13.4 Conclusion 143
Acknowledgments 144
References 144
14 Proteins of Coxiella burnetii and Analysis of Their Function 145
Robert Ihnatko, Pavol Vadovicˇ, and Rudolf Toman
14.1 Introduction 145
14.2 Proteins of C. burnetii and Their Functions 146
14.3 Conclusion and Perspectives 149
Note Added in Proof 149
Acknowledgments 150
References 150
15 Subtype and Toxin Variant Identification of Botulinum Neurotoxin Type A Using Proteomics Techniques 153
Suzanne R. Kalb, Jakub Baudys, Theresa J. Smith, James L. Pirkle, and John R. Barr
15.1 Introduction 153
15.2 Botulinum Neurotoxins 153
15.3 Differentiation of Botulinum Neurotoxins 154
15.4 Amino Acid Sequence Identifi cation Using Proteomics 155
15.5 Extraction of BoNT from Complex Matrices 157
15.6 Identifi cation of BoNT Serotype with Proteomics 157
15.7 Identifi cation of BoNT/A Subtype with Proteomics 159
15.8 Identifi cation of BoNT/A1 Strain with Proteomics 161
15.9 Conclusions 163
Disclaimer 164
References 164
16 Protein Microarrays for Antigen Discovery 167
Mohan Natesan, Sarah Keasey, and Robert G. Ulrich
16.1 Introduction 167
16.2 Microarray Assembly 168
16.3 Antibody Assays 170
16.4 Antigens and Proteomes of Viruses 171
16.5 Antigens and Proteomes of Pathogenic Bacteria 174
16.6 Conclusions 176
Acknowledgment 177
References 177
17 MALDI–TOF Mass Spectrometry for Rapid Identification of Highly Pathogenic Microorganisms 179
Peter Lasch and Dieter Naumann
17.1 Introduction 179
17.2 Microbial Identification by MALDI–TOF Mass Spectrometry 180
17.3 Inactivation of Highly Pathogenic Microorganisms for MALDI–TOF Mass Spectrometry 187
17.4 Identification of Important Bacterial Pathogenes Using MALDI–TOF MS 192
17.5 Concluding Remarks 207
Acknowledgments 208
References 208
Part Three Analysis of Host–Pathogen Interactions 213
18 Quantitative Proteomic Profiling of the Interaction of Francisella tularensis LVS with Macrophages Using J774.2 Cell Line 215
Anetta Hartlova, Marek Link, Juraj Lenco, and Jiri Stulik
18.1 Introduction 215
18.2 Material and Methods 216
18.3 Results 218
References 221
19 Proteome Analysis of Bacterial Protein Expression after Ingestion of Microbes by Macrophages 223
Martin Brychta and Ivona Pávková
19.1 Intro duction 223
19.2 Material and Methods 224
19.3 Results 226
19.4 Discussion 228
Acknowledgment 230
References 230
Index 233

Nota biograficzna:
Prof. J. Stulik, MD, PhD is a head of the Institute of Molecular Pathology, Faculty of Military Health Sciences. This institute is the only facility in Czech Republic engaged in biodefense research. The research activity is focused on intracellular pathogen Francisella tularensis that is classified as a class A agent by the U.S. government. The institute exploits the methods of functional genomics and system biology for the identification of both microbial and host molecules involved in host–pathogen interaction. The results of this study are important for development of new prophylactic and therapeutic agents against tularemia.
Rudolf Toman, PhD, DSc is head of the Laboratory for Diagnosis and Prevention of Rickettsial and Chlamydial Infections at the Institute of Virology of the Slovak Academy of Sciences in Bratislava, Slovakia. The laboratory is engaged in microbiological and glycomics/proteomics studies of BSL3 bacteria that potentially can be used as biological warfare agents. The special emphasis is put on the intracellular pathogen Coxiella burnetii that is classfied as a category B biological warfare agent by the US authorities. Structure/function relationship studies of the surface macromolecules (lipopolysaccharides and proteins) of C. burnetii, Rickettsiae and Chlamydiae are performed together with characterization of their immunodominant epitopes with the aim to develop new diagnostic and prophylactic agents against these highly infectious pathogens.
Patrick Butaye is a veterinary researcher at the Veterinary and Agrochemical Research Center in Brussels with a focus on antimicrobial resistance in bacteria from animal origin.
Robert G. Ulrich is chief of the Department of Immunology and head of the Laborato ry of Molecular Immunology at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). USAMRIID is the primary U.S. Department of Defense laboratory dedicated to research in biodefense and infectious diseases. He received a Ph.D. in Microbiology and Immunology from Baylor College of Medicine. His laboratory uses microarrays and other proteomic tools to study virulence factors, protein interaction networks and host responses to infectious diseases. This work has resulted in the development of many patented diagnostic assays and vaccines.