Implication of extraintestinal pathogenic Escherichia coli siderophore receptors in host pathogen interaction [Elektronische Ressource] / Friederike Feldmann

Rike

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 18
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
Lehrstuhl für Mikrobiologie



Implication of extraintestinal pathogenic Escherichia coli
siderophore receptors in host pathogen interaction



Friederike Feldmann



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. W. Liebl
Prüfer der Dissertation: Univ.-Prof. Dr. K.-H. Schleifer (i. R.)
Priv.-Doz. Dr. S. Schubert,
(Ludwig-Maximilians-Universität München)



Die Dissertation wurde am 30.04.2008 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 16.07.2008 angenommen.





























meiner Familie






Table of contents

TABLE OF CONTENTS
TABLE OF CONTENTS......................................................................................................... 5
ABBREVIATIONS .................................................................................................................. 9
1. INTRODUCTION......................................................................................................... 13
1.1. The species Escherichia coli ........................................................................................ 13
1.2. Extraintestinal pathogenic Escherichia coli (ExPEC) ................................................. 16
1.3. Outer membrane vesicles ............................................................................................. 29
1.4. Urinary tract infections................................................................................................. 30
1.5. Host pathogen interaction and modulation of immune response ................................. 32
1.6. Aims of the study ......................................................................................................... 35
2. MATERIALS................................................................................................................. 37
2.1. Strains........................................................................................................................... 37
2.2. Cell culture collection .................................................................................................. 39
2.3. Cell culture media and solutions .................................................................................. 39
2.4. Plasmids ....................................................................................................................... 40
2.5. Oligonucleotides........................................................................................................... 41
2.6. Chemicals and Enzymes............................................................................................... 45
2.7. Antibodies .................................................................................................................... 46
2.8. DNA and Protein Markers............................................................................................ 47
2.9. Inhibitors ...................................................................................................................... 47
5 Table of contents

2.10. Media, agar plates and antibiotics................................................................................ 48
2.11. Technical Equipment.................................................................................................... 50
3. METHODS .................................................................................................................... 51
3.1. Bacterial strains ............................................................................................................ 51
3.2. Cell culture ................................................................................................................... 51
3.3. Manipulation of DNA .................................................................................................. 55
3.4. Manipulation of RNA................................................................................................... 61
3.5. Working with Protein................................................................................................... 63
3.6. Isolation of outer membrane proteins (OMPs) (modified from Hantke, 1981) ........... 69
3.7. Isolation of bacterial outer membrane vesicles (OMVs) ............................................. 70
3.8. Isolation of lipopolysaccharides (LPS) ........................................................................ 71
3.9. Microscopy................................................................................................................... 72
3.10. Assay of hemolytic activity.......................................................................................... 73
3.11. Cytokine ELISAs ......................................................................................................... 73
4. RESULTS....................................................................................................................... 75
4.1. Prevalence of ExPEC virulence factors ....................................................................... 75
4.2. Characterization of the siderophore receptors FyuA and IroN .................................... 78
4.3. Functional relevance of FyuA and IroN: invasion assays............................................ 84
4.4. Outer membrane vesicles of ExPEC ............................................................................ 95
4.5. Hemolytical active α-hemolysin is present in ExPEC vesicles.................................. 105
4.6. Influence of ExPEC vesicles on the cytokine profile of urothelial cells.................... 119
6 Table of contents

5. DISCUSSION .............................................................................................................. 123
5.1. Expression and subcellular localization of FyuA and IroN ....................................... 123
5.2. Functional relevance of FyuA and IroN..................................................................... 125
5.3. IroN vesicles associate with urothelial cells .............................................................. 129
5.4. ExPEC vesicles and α-hemolysin .............................................................................. 132
5.5. Immunological role of ExPEC vesicles ..................................................................... 136
6. SUMMARY.................................................................................................................. 139
7. ZUSAMMENFASSUNG ............................................................................................ 141
8. REFERENCES............................................................................................................ 143
9. APPENDIX .................................................................................................................. 165
9.1. List of original publications ....................................................................................... 165
9.2. Oral presentation ........................................................................................................ 166
9.3. Poster presentations.................................................................................................... 167
9.4. Curriculum vitae......................................................................................................... 169
9.5. Acknowledgements .................................................................................................... 173

7

8 Abbreviations

ABBREVIATIONS
AA amino acid
Afa AfaDE family of adhesins
Afa/Dra Afa/Dr family of adhesins
Auf Auf fimbriae
cDNA complementary DNA
CDT cytolethal distending toxin
CNF cytotoxic necrotizing factor
ChuA outer membrane hemin receptor
Cva colicin
°C degree celsius
DABCO 1,4-diazabicyclo-(2.2.2)octane
DAPI 4’6’-diamidino-2-phenylindole hydrochloride
DNA desoxyribonucleicacid
dH O distilled water 2
Dra Dr fimbriae
DTT dithio…
ELISA enzyme linked immunosorbent assay
Ent enterobactin siderophore system
EPEC enteropathogenic E. coli
ExPEC extraintestinal pathogenic E. coli
FACS fluorescence activated cell sorting
FepA siderophore enterobactin receptor
Fim type I fimbriae
FITC fluorescein isothiocyanate
Foc F1C fimbriae
FyuA siderophore yersiniabactin receptor
Hbp hemoglobin-protease
Hek hemagglutinin from E. coli K1
HlyA alpha-hemolysin
Hra heat-resistant agglutinin
Iha IrgA-homologue adhesin
IC intermediate cell
9 Abbreviations

Ig immunoglobulin
IL interleukin
IPTG isopropyl-β-D-thi