CEACAM3-mediated phagocytosis of human-specific bacterial pathogens involves the adaptor molecule Nck [Elektronische Ressource] / vorgelegt von Lisa Peterson

Lpeterso

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Aus dem Institut für Molekulare Infektionsbiologie/
Zentrum für Infektionsforschung
der Universität Würzburg
Vorstand: Professor Dr. med. Matthias Frosch
Ehem. Vorstand: Professor Dr. Dr. h. c. mult. Jörg Hacker



CEACAM3-mediated phagocytosis of human-specific bacterial
pathogens involves the adaptor molecule Nck



Inaugural - Dissertation
zur Erlangung der Doktorwürde der
Medizinischen Fakultät
der
Julius-Maximilians-Universität zu Würzburg
vorgelegt von
Lisa Peterson
aus Hannover

Würzburg, Dezember 2008 Referent: Professor Dr. Dr. h. c. mult. Jörg Hacker
Koreferent: Professor Dr. med. Dr. rer. nat. Bhanu Sinha

Dekan: Professor Dr. med. Matthias Frosch


















Tag der mündlichen Prüfung: 17.12.2009

Die Promovendin ist Ärztin.














Meinen Eltern.
Contents
1 INTRODUCTION......................................................................................... 1
1.1 Neisseria gonorrhoeae.......................................................................... 2
1.1.1 Clinical picture .................................................................................. 3
1.1.2 Adhesion and invasion - molecular processes of infection ............... 3
1.1.3 The pilus........................................................................................... 5
1.1.4 Opacity proteins................................................................................ 6
1.2 Interaction of gonococci with epithelia and endothelia ..................... 8
1.2.1 Interaction of Opa-proteins with heparansulphate-proteoglycanes... 8
1.2.2 Interaction of Opa-proteins with CEACAM molecules .................... 10
1.3 Interaction of gonococci with the human immune system.............. 15
1.3.1 Interaction of N.gonorrhoeae with elements of adaptive immunity . 15
1.3.2 Antigenic and phase variation ........................................................ 17
1.3.3 IgA protease ................................................................................... 19
1.3.4 Interaction with T-lymphocytes and immunosuppression via ITIM . 20
1.3.5 Interaction of N.gonorrhoeae with elements of innate immunity ..... 21
1.3.6 The opsonin-independent uptake of gonococci .............................. 22
1.3.7 The oxidative burst induced by Opa -carrying gonococci ........... 24 CEA
1.4 CEACAM3............................................................................................. 24
1.4.1 The role of the ITAM-motif in CEACAM3........................................ 25
1.4.2 Molecular events following CEACAM3 receptor engagement ........ 27
1.5 Aims and objectives............................................................................ 29
2 MATERIALS ............................................................................................. 32
2.1 Bacteria ................................................................................................ 32
2.1.1 Neisseria ........................................................................................ 32
2.1.2 E.coli............................................................................................... 32
2.2 Cell lines............................................................................................... 32
2.3 Culture media....................................................................................... 32
2.3.1 Bacterial culture.............................................................................. 32
2.3.2 Cell culture ..................................................................................... 33
2.4 Antibodies ............................................................................................ 33
2.4.1 Anti-CEACAM................................................................................. 33
2.4.2 Anti-N.gonorrhoeae ........................................................................ 34
2.4.3 Other .............................................................................................. 34
2.4.4 Secondary antibodies..................................................................... 34 2.5 Enzymes and proteins......................................................................... 34
2.5.1 Enzymes......................................................................................... 34
2.5.2 Proteins .......................................................................................... 34
2.6 Plasmids and oligonucleotides .......................................................... 35
2.6.1 Plasmids......................................................................................... 35
2.6.2 Oligonucleotides............................................................................. 35
2.7 Buffers and solutions.......................................................................... 36
2.7.1 For eukaryotic cells......................................................................... 36
2.7.2 Molecular biology............................................................................ 36
2.7.3 SDS-PAGE, Coomassie stain, Western blot .................................. 37
2.7.4 Protein purification.......................................................................... 38
2.8 Kits and reagents................................................................................. 38
2.9 Laboratory tools .................................................................................. 39
3 METHODS ................................................................................................ 40
3.1 Handling of bacteria ............................................................................ 40
3.1.1 Bacterial culture.............................................................................. 40
3.1.2 Storage........................................................................................... 40
3.1.3 Selection......................................................................................... 40
3.1.4 Rendering bacteria competent........................................................ 41
3.1.5 Transformation of bacteria.............................................................. 41
3.2 Handling of eukaryotic cells ............................................................... 41
3.2.1 Cell culture ..................................................................................... 41
3.2.2 Storage of eukaryotic cells ............................................................. 42
3.2.3 Cell count ....................................................................................... 42
3.2.4 Transfection of cells........................................................................ 42
3.2.5 Cell lysates ..................................................................................... 43
3.2.6 Isolation of primary human granulocytes........................................ 43
3.3 Protein handling .................................................................................. 44
3.3.1 Denaturing sodiumdodecylsulfate-polyacrylamide gel
electrophoresis ............................................................................... 44
3.3.2 Western blotting.............................................................................. 44
3.3.3 Coomassie staining ........................................................................ 45
3.3.4 Protein purification.......................................................................... 45
3.4 Handling of DNA .................................................................................. 46
3.4.1 Agarose gel electrophoresis ........................................................... 46
3.4.2 DNA preparation from agarose gels ............................................... 46
3.4.3 DNA preparation from bacteria (Miniprep, Midiprep) ...................... 47
3.4.4 Polymerase chain reaction ............................................................. 48
TM3.4.5 PCR-Cloning using the InFusion-Reaction .................................. 49
TM3.4.6 Cloning using the Cre-loxP Site-Specific Recombination ............ 50 3.4.7 In vitro site-specific mutagenesis.................................................... 52
3.4.8 Restriction digest............................................................................ 53
3.4.9 DNA-sequencing ............................................................................ 53
3.5 In vitro protein interaction studies..................................................... 54
3.5.1 GST-pull down assay ..................................................................... 54
3.5.2 Co-immunoprecipitation.................................................................. 55
3.6 Infection studies .................................................................................. 55
3.6.1 Gentamicin protection assay .......................................................... 55
3.6.2 Immunofluorescence studies.......................................................... 56
3.6.3 Confocal laser-scanning microscopy.............................................. 56