Listeria monocytogenes as a vaccine vehicle [Elektronische Ressource] : generation of attenuated mutants and their immunological characterization / submitted by Walid Kamal Abdel-Naby Mohamed
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Listeria monocytogenes as a vaccine vehicle [Elektronische Ressource] : generation of attenuated mutants and their immunological characterization / submitted by Walid Kamal Abdel-Naby Mohamed

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Learn all about the services we offer
216 Pages
English

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Listeria monocytogenes as a vaccine vehicle: generation of attenuated mutants and their immunological characterization A thesis submitted in partial fulfillments of the requirements for the degree of Doctor in Human Biology (Dr. biol. hom.) in the faculty of medicine at Justus-Liebig-University Giessen Submitted by Walid Kamal Abdel-Naby Mohamed Sohag, Egypt Gießen (2004) From Institute of Medical Microbiology Director: Prof. Dr. Trinad Chakraborty Faculty of medicine Justus-Liebig-University Giessen 1. Advisor: Prof. Dr. T. Chakraborty 2. Advisor: PD. Dr. H. Hackstein Date of disputation: 29. 03. 2005 List of Original Publications Part of this work is based on the following original publications that are reproduced with the permission of the publishers. Darji, A., Mohamed, W., Domann, E. and Chakraborty, T. (2003) Induction of immune responses by attenuated isogenic mutant strains of Listeria monocytogenes. Vaccine 21, 102-129. Mohamed, W., Darji, A., Domann, E., Chiancone, E. and Chakraborty, T. The ferritin protein Frm, a novel listerial antigen, mediates hydrogen peroxide resistance and is required for efficient intracellular growth of Listeria monocytogenes. Submitted for publication. Mohamed, W., Darji, A. and Chakraborty, T.

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Published 01 January 2005
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Listeria monocytogenes as a vaccine vehicle:
generation of attenuated mutants and their
immunological characterization






A thesis submitted in partial fulfillments of the requirements for the
degree of Doctor in Human Biology (Dr. biol. hom.) in the faculty of
medicine at Justus-Liebig-University Giessen





Submitted by
Walid Kamal Abdel-Naby Mohamed
Sohag, Egypt



Gießen (2004)



From Institute of Medical Microbiology
Director: Prof. Dr. Trinad Chakraborty
Faculty of medicine
Justus-Liebig-University Giessen















1. Advisor: Prof. Dr. T. Chakraborty

2. Advisor: PD. Dr. H. Hackstein

Date of disputation: 29. 03. 2005



List of Original Publications


Part of this work is based on the following original publications that are reproduced with
the permission of the publishers.

Darji, A., Mohamed, W., Domann, E. and Chakraborty, T. (2003) Induction of
immune responses by attenuated isogenic mutant strains of Listeria monocytogenes.
Vaccine 21, 102-129.

Mohamed, W., Darji, A., Domann, E., Chiancone, E. and Chakraborty, T. The
ferritin protein Frm, a novel listerial antigen, mediates hydrogen peroxide resistance and
is required for efficient intracellular growth of Listeria monocytogenes. Submitted for
publication.

Mohamed, W., Darji, A. and Chakraborty, T. The PEST-like region in Listeriolysin-
O is critical for induction of effective long-term immunity. Submitted for publication.





















To my family Table of Contents I
Table of Contents
I
List of Abbreviations VII
1. Introduction 1
1.1. Problems of infectious diseases 1
1.2. Vaccines 2
1.2.1. Virus-based vaccine vectors 4
1.2.2. Bacterial-based vaccine vectors 5
1.2.2.1. Listeria vector vaccines 6
1.2.2.1.1. Listeria delivery of plasmid DNA 8
1.2.3. Vaccines and T cells 8
1.2.3.1. Stages of T-cell responses 9
1.2.3.2. T-cell differentiation 9
1.2.3.3. T cell migration 10
1.2.3.4. Lineage of memory T cells 11
1.2.3.5. Immunological characters of naive versus memory T cells 12
1.3. Interactions of Listeria monocytogenes with mammalian host cells and tissues 13
1.3.1. Entry into cells 14
1.3.2. Escape from a vacuole 15
1.3.3. Compartmentalization of LLO activity 16
1.3.4. Growth in the cytosol 17
1.3.5. Cell to cell spread 18
1.3.6. The virulence gene cluster of L. monocytogenes 19
1.3.7. Listeriosis model of systemic infections 21
1.4. Host immune response to Listeria infection 23
1.4.1. The innate response to Listeria infection 23
1.4.2. The adaptive response to Listeria infection 25
1.4.2.1. The T cell response 25
1.4.2.1.1. CD8+ T cell effector mechanisms 26
d1.4.2.1.2. H2-K -restricted recognition by T cells 26
1.4.2.1.3. H2-M3-restricted recognition by T cells 27
1.4.2.2. Role of humoral immunity in Listeria infection 28 Table of Contents II
1.5. Cholesterol-binding cytolytic protein toxins 29
1.5.1. Pneumolysin 30
1.5.1.1. Immunomodulatory effects of pneumolysin 32
1.6. Regulation of iron uptake and storage in L. monocytogenes 33
1.7. Aim of this work 35
2. Materials and Methods 37
2.1. Bacterial strains and plasmid vectors 37
2.2. Chemicals and biochemicals 39
2.3. Culture media, supplements, buffers and solutions 39
2.3.1. Culture media 39
2.3.2. Media supplements 41
2.3.3. Buffers and solutions 42
2.4. Bacterial storage 48
2.5. Bacterial growth conditions 48
2.6. Bacterial growth measurement 48
2.7. Molecular biological and molecular genetics methods 49
2.7.1. DNA isolation 49
2.7.1.1. Plasmid DNA Isolation from E. coli 49
2.7.1.2. Chromosomal DNA isolation from gram-positive bacteria 49
2.7.2. Enzymatic treatment of DNA 50
2.7.2.1. DNA digestion with restriction enzyme 50
2.7.2.2. Ligation of a DNA fragment with a DNA vector 50
2.7.3. Agarose gel electrophoresis 50
2.7.4. Extraction of DNA fragments from the agarose gel 51
2.7.5. Quantification of DNA concentration 51
2.7.6. Transformation 52
2.7.6.1. Transformation in E. coli 52
2.7.6.1.1. CCMB80 method 52
2.7.6.2. Transformation in L. monocytogenes by electroporation 52
2.7.7. Polymerase chain reaction (PCR) 53
2.7.7.1. Amplification of DNA fragments for further cloning 53
2.7.7.2. Amplification of DNA fragments for testing the recombinant clone 54
2.7.8. Construction of a site-directed insertion mutation 55
2.7.9. DNA sequencing 57 Table of Contents III
2.7.10. Computer programs 57
2.7.11. Primers 58
2.8. Protein biochemical methods 58
2.8.1. Protein isolation from Listeria species 58
2.8.1.1. Proteins in bacterial supernatant 58
2.8.1.2. Somatic soluble antigens 59
2.8.2. Protein analysis 59
2.8.2.1. SDS-Polyacrylamide Gel Electrophoresis 59
2.8.2.2. Immunoblotting (Western blot) 60
2.8.2.2.1. BCIP immunodetection procedure 60
2.8.2.2.2. Enhanced chemiluminescence (ECL) immunodetection procedure 61
2.8.3. Protein Purification 61
2.8.4. Screening of hemolytic activity 63
2.9. Cell culture 63
2.9.1. Eukaryotic cell lines 63
2.9.2. Cell culture media and supplements 64
2.9.3. Counting the eukaryotic cells using a microscope counting chamber 65
2.9.4. Culture of eukaryotic cell 66
2.9.5. Storage of eukaryotic cells 66
2.9.6. Infection of eukaryotic cell lines with Listeria strains 67
2.9.6.1. Invasion assay 67
2.9.6.2. Plaque assay 68
2.9.6.3. Immunofluorescence microscopy 69
2.10. Immunological methods 70
2.10.1. Experimental mice infection 70
2.10.2. Determination of bacterial load in infected organs 70
2.10.3. Production of protein-specific antibodies 71
2.10.3.1. Polyclonal antibodies 71
2.10.3.2. Monoclonal antibodies (mAb) 71
2.10.4. Detection of Listeria-specific antibodies 73
2.10.5. DTH response to somatic listerial antigen 74
2.10.6. Stimulation of spleen cells in vitro for cytokine production 75
2.10.7. Cytokines ELISA 75
2.10.8. Cytokines ELISPOT 76 Table of Contents IV
2.10.9. Flow Cytometry Analysis 77
3. Results 79
3.1. Purification and characterization of thiol-activated cytolysins hyper-expressed
in the non-pathogenic species Listeria innocua 79
79 3.1.1. Listeriolysin O
80 3.1.1.1. Bacterial strain and growth conditions
81 3.1.1.2. Purification and characterization of listeriolysin O
82 3.1.2. Pneumolysin
3.1.2.1. Bacterial strain and growth conditions 83
3.1.2.2. Purification and characterization of pneumolysin 83

3.2. The ferritin protein Frm, a novel listerial antigen, mediates hydrogen peroxide
resistance and is required for efficient intracellular growth of L. monocytogenes 85
3.2.1. Bacterial strains and culture 85
3.2.1.1. Generation of the ∆frm mutant and its complementation 86
3.2.2. Detection of Frm during infection 87
3.2.3. Properties of the ∆frm L. monocytogenes mutant 88
3.2.4. Frm mediates resistance to the effects of hydrogen peroxide. 92
3.2.5. Frm promotes bioaccessibility of mineralized iron 92
3.2.6. The ∆frm mutant exhibits defects at early stages of infection 93
3.3. Induction of immune responses by attenuated isogenic mutant strains of
95 Listeria monocytogenes
3.3.1. Survival and persistence of wild type L. monocytogenes and isogenic
96 L. monocytogenes mutant strains in vivo
3.3.2. Spleen morphology on day 4 post-infection 97
3.3.3. Listeria-induced IFN- γ production of spleen cells in vitro and DTH-
98 response in vivo
3.3.4. Acquired protection conferred by isogenic attenuated mutants 99
3.3.5. Induction of listeriolysin O-specific antibodies against isogenic
103 L. monocytogenes deletion mutant strains
3.4. Immunological characterization of a L. innocua recombinant strain carrying
105 the virulence gene cluster (vgc) of L. monocytogenes
3.4.1. Growth kinetics of the recombinant L. innocua strain in vivo 105Table of Contents V
3.4.2. Downmodulation of CD4+-mediated inflammatory responses by the
recombinant L. innocua derivative 106
3.4.3. Expression of T cell-mediated immune response to the recombinant L.
innocua strain 108
3.4.4. Induction of listeriolysin O specific antibody in response to the
recombinant L. innocua: :vgc strain. 111
3.5. Molecular and immunological characterization of a Listeria monocytogenes
strain harbouring a gene of pneumolysin in instead of listeriolysin O 114
3.5.1. Bacterial strains and growth conditions 114
3.5.2. Construction of plasmid-based strains 115
1153.5.2.1. EGD-e ∆hly::pSOG306-ply
3.5.2.2. EGD-e ∆hly::pSOG304-hly 116
3.5.3. Expression and hemolytic activity of pneumolysin 116
3.5.4. Listeria expressing pneumolysin shows a limited intracellular growth in
J774 macrophages 117
3.5.5. L. monocytogenes expressing pneumolysin shows a reduced in vivo
118 survival
3.5.6. Induction of a protective antilisterial immune response by Listeria
119 monocytogenes expressing pneumolysin in place of listeriolysin O
3.5.7. Humoral response is induced against both listeriolysin O
124 and pneumolysin
3.6. The PEST-like region in Listeriolysin O is critical for induction of
126 effective long-term immunity
3.6.1. Generation of the LLO mutant protein 126
3.6.1.1. Bacteria 126
3.6.1.2. Construction of the mutants 127
1273.6.1.2.1. EGDe ∆hly::pSOG304-hly ∆PEST
1273.6.1.2.2. EGD-e ∆hly: :pSOG304-hly
3.6.2. Expression and hemolytic activity of the LLO mutant protein 128
3.6.3. Deletion of the PEST-like sequence of LLO inhibits bacterial phagosomal
129 escape
3.6.4. PEST-like sequence mediates survival of L. monocytogenes in vivo 131
3.6.5. Spleen morphology on day 3 after infection 132
3.6.6. PEST-like sequence truncated Listeriolysin O fails to induce IFN- γ either
133 in serum or by splenocytes during primary infection
3.6.7. Expression of acquired immunity by the wild type L. monocytogenes and its
134 isogenic derivatives Table of Contents VI
4. Discussion 138

4.1. Antibody responses are important in defense during infection with intracellular
138 bacteria
4.2. Tailored bacterial vehicles as vaccine strains 144
4.3. Listeriolysin O is not absolutely essential for induction of long term
cellular immunity against Listeria monocytogenes 150
4.4. Cytosolic localisation of L. monocytogenes is critical for induction of
154 protective immunity
4.5. Outlook 158
4.5.1. Antibody-mediated immunity against intracellular pathogens 158
4.5.2. Requirements for the creation of novel vaccine vectors 158
4.5.3. Role of Listeriolysin O in Listeria monocytogenes infection 160
5. Summary 161
6. Zusammenfassung 164
7. References 167