NF-kappaB activation in infections with Helicobacter pylori or Legionella pneumophila [Elektronische Ressource] / von Sina Bartfeld

Sina Bartfeld

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2009
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation
NF-kappaB activation in infections
with Helicobacter pylori
or Legionella pneumophila
zur Erlangung des akademischen Grades doctor rerunma turalium (Dr. rer. nat)
im Fach Biologie
eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt Universität zu Berlin
von Dipl.-Biol. Sina Bartfeld (geb. 06.01.1e9r7li8n )i n B
Präsident der Humboldt Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Faku ltät I
Prof. Dr. Lutz-Helmut Schön


Gutachter: 1. Prof. Thomas F. Meyer
2. Prof. Wolfgang Uckert
3. Prof. Claus Scheidereit

Tag der mündlichen Prüfun: g30.6.2009
Table of content

Table of conten t
Abbreviations ........................................................ .. .4.............
Abstract ....................................................................................... ...... .6..........
Zusammenfassung ................................................... .7..................
1 Introduction ...................................................... .8................
1.1 The transcription factor family NF-κB ......................... .................... 9
1.2 The bacterium Helicobacter pylor i.................................... .... .1.6................
1.3 The intracellular bacteriumLe gionella pneumophila ................. ............. 21
1.4 RNAi-based screens ........................................3. ....................... 2
1.5 Aims of this thesis ........................................ .2 4.......................
2 Results and Discussion ............................................. ................. 25
2.1 Part I: Imaging NF-κB activation using p65-.GF.P. .................... ............... 25
2.1.1 A high throughput assay for cell lines expressing6 5p-GFP ....... .. 25
2.1.2 Comparison of reporter cell lines and parenta ll icneells ....... ..... 27
2.1.3 Specific temporal control of p65-GFP translocati o.n.s......... ....... 28
2.1.4 Bacterial infection induces oscillations of p6 5.-.G.FP......... ......... 29
2.1.5 Discussion............................................ .................... 32
2.2 Part II: RNAi-based screen identified regulat oNFr-s κBo fsignaling ..... .. 35
2.2.1 Establishment of the RNAi-based screen................... .......... 35
2.2.2 Performance of the screen ............................. .............. 36
2.2.3 Quality control of the screen and identificati ohnit so f......... ..... 37
2.2.4 Validation of identified genes ........................ ............... 37
2.2.5 Time resolved analysis of NF-κB regulators ............... .......... 40
2.2.6 Combinatorial effects of identified siRNAs. ...................... ........... 43
2.2.7 ALPK1 and CRKRS are important for NF-κB activati.o.n. ....... ...... 45
2.2.8 SKP2 is necessary for termination of NF-κB activ .it.y.......... ........ 48
2.2.9 Discussion............................................ .................... 51
2.3 Part III: Two tracks of NF-κB activation indu Lce. dp nbeyumophila ..... .. 59
2.3.1 Biphasic p65 translocation duriLn.g pneumophila infection .... .. 59
2.3.2 NF-κB activation profiles oLf. pneumophila wild-type strains .. .. 60
2.3.3 Transient p65 translocation depends on intactl laf la.g.e....... ..... 60
2.3.4 Continuous p65 translocation depends on the Dot /sIycsmtem. 6 2
2.3.5 Involvement of single Dot/Icm secreted effec.to.r.s. ........ ........ 62
2.3.6 Bacterial replication and continuous p65 transilocna t ........ ..... 63
2.3.7 IκBα is degraded during continuous activatio.n. ........... ......... 64
2.3.8 TLR5 and MyD88 are only important for early aicotinv a.t...... .... 65
2.3.9 Discussion................................................................. .................... 66
3 Conclusions and Outlook .......................................... ................. 70
4 Material and Methods .............................................. ................. 73
4.1 General suppliers ......................................... .7. 3.......................

2Table of content

4.2 DNA-constructs and molecular biology techniques ................. ............ 73
4.4 Lentiviral transduction and generation of monol clcoenlla lines ....... ..... 75
4.5 Transient transfection of siRNA .............................. .................... 76
4.6 Automated transient transfection of siRNA ..................... ............... 76
4.7 Bacterial culture ................................................................ . 7.7.....................
4.8 Infections ........................................................................... .. .7.8..................
4.9 Intracellular growth ofL . pneumophila ......................... .7. 8.......................
4.10 Automated microscopy and software-based picture lyasnias .... 78
4.11 Live cell microscopy .................................. ................. 80
4.12 Immunofluorescence after live cell microscopy. ............ ........ 81
4.13 SDS-PAGE ................................................................. .................... 81
4.14 Immunoblotting .......................................................... ................. 81
4.15 Kinase assay ......................................... .................... 82
4.16 ELISA ............................................... ...................... 83
4.17 qRT-PCR ............................................ ..................... 83
5 Statistics ......................................................... .8..5.............
5.1 Statistical analysis of live cell microscopy. ............................ .................. 85
5.2 Statistical analysis of screen and hit valida.ti.o.n. ................. ................. 85
6 References ...................................................... .9. .2................
Publications ....................................................... ..1 .0.4.............
Selbstständigkeitserklärung .....................................................5. ................ 104
Acknowledgements .................................................0.6. ................. 1


3Abbreviations

Abbreviations
AICD activation induced cell death IL interleukin
ALPK1 alpha kinase 1 ILK1 integrin-linked protein kinase 1
APS ammonium peroxodisulfate IRAK interleukin-1 receptor-associated kinase
AURKB aurora/IPL1-related kinase 2 kDa kilodalton
BAFF B cell-activating factor LB Luria Bertani
Bcl-3 B cell lymphoma 3 LCV Legionella containing vacuole
βTrCP β transducin repeat-containing protein LetA Legionella transmission activator LetA
CagA cytotoxin-associated gene A LPS lipopolysaccharide
CCNB1 G2/mitotic-specific cyclin B1 LT lymphotoxin
CCNB2 G2/mitotic-specific cyclin B2 LTK leukocyte tyrosine kinase
CD40 cluster of Differentiation 40 LubX Legionella U-box containing protein LubX
CDC cell division cycle MALT mucosa-associated lymphatic tissue
CDK cyclin dependent kinase MAMP microbe associated molecular pattern
Cezanne cellular zinc finger anti NF-κB MAPK mitogen-activated protein kinase
c-FLIP Fas-associated via death domain (FADD)- MEF mouse embryonic fibroblasts
like interleukin-1-β-converting enzyme MEKK mitogen-activated protein/ERK kinase
(FLICE/caspase 8)-inhibitory protein kinase
CFP cyan fluorescent protein MHCK A myosin heavy chain kinase a
CFU colony forming unit MIP-2 macrophage inflammatory protein 2
CHED cholinesterase-related cell division con- MOI multiplicity of infection
troller mRNA messenger RNA
c-IAP inhibitor of apoptosis protein NOD nucleotide binding and oligomerization
Clk1 CDC-like kinase 1 domain
c-Rel v-rel reticuloendotheliosis viral oncogene p100 NF-κB subunit p100
homolog p105 NF-κB subunit p105
CRKRS CDC2-related kinase, arginine/serine rich p50 NF-κB subunit p50
CYLD cylindromatosis protein p52 NF-κB subunit p52
DMEM Dulbecco’s modified eagle medium p65 NF-κB subunit p65
DMSO dimethyl sulfoxide PAA polyacrylamide
DNA desoxyribonucleic acid PAGE polyacrylamide gel electrophoresis
Dot defective in organelle trafficking PAI pathogenicity island
DUB de-ubiquitinylation PAK1 p21 protein (CDC42/Rac)-activated kinase
DYRK1B dual-specificity tyrosine-phosphorylation 1
regulated kinase 1B PCR polymerase chain reaction
ECL enhanced chemiluminescence PG peptidoglycan
EGF epidermal growth factor PLK1 polo-like kinase 1
EGFR epidermal growth factor receptor PRKACB cAMP-dependent protein kinase, β-
ELKS protein rich in amino acids E, L, K and S catalytic subunit
EMSA electro mobility shift assay PRKCABP PRKCA-binding protein
EnhC Legionella enhanced entry protein EnhC PRKD2 protein kinase C, D2 type
ERK extracellular signal-regulated kinase PRR pattern recognition receptor
FASTK FAS-activated serine/threonine kinase PSKH1 serine/threonine-protein kinase H1
FCS fetal calf serum PtsP Legionella phosphoenolpyruvate protein
FGFR4 fibroblast growth factor receptor 4 phosphotransferase PtsP
FlaA Legionella flagellin PVDF polyvinylidene fluoride
FliA Legionella flagellar biosynthesis sigma RelA v-rel reticuloendotheliosis viral oncogene
factor FliA homolog A
GAK cyclin G-associated kinase RelB v-rel reticuloendotheliosis viral oncogene
GAPDH glyceraldehyde 3-phosp