Characterization of type III secreted bacterial virulence factors that interfere with Rho GTPase signalling

Bakterien, wie Salmonella enterica, Shigella flexneri oder pathogenen Escherichia coli verursachen Infektionskrankheiten. Um eine bakterielle Infektion zu etablieren, manipulieren Bakterien mit Hilfe von eingeschleusten Virulenzfaktoren die Signalwege der Wirtszelle. Im Rahmen dieser Arbeit habe ich...

Other title:Characterization of type-III secreted bacterial virulence factors that interfere with Rho-GTPase signalling
Author: Arens, Stefan
Further contributors: Stradal, Theresia (Thesis advisor)
Division/Institute:FB 13: Biologie
Document types:Doctoral thesis
Media types:Text
Publication date:
Date of publication on miami:07.01.2014
Modification date:27.07.2015
Edition statement:[Electronic ed.]
Subjects:WxxxE; EPEC; Infektion; Virulenzfaktoren; RHPN; Map
DDC Subject:570: Biowissenschaften; Biologie
License:InC 1.0
Language:English
Format:PDF document
URN:urn:nbn:de:hbz:6-74359366333
Permalink:http://nbn-resolving.de/urn:nbn:de:hbz:6-74359366333
Digital documents:diss_arens_stefan.pdf
Table of contents:
  • Content
  • 1 Introduction .......................................................................................................... 1
  • 1.1 Host pathogen interaction ............................................................................. 1
  • 1.2 The Host ........................................................................................................ 2
  • 1.2.1 Motion .................................................................................................... 2
  • 1.2.1.1 Lamellipodia and ruffles ................................................................. 2
  • 1.2.1.2 Filopodia and Microspikes ............................................................. 5
  • 1.2.1.3 Stress fibers .................................................................................... 6
  • 1.2.1.4 Small GTPases ................................................................................ 8
  • 1.2.1.5 Actin polymerization .................................................................... 10
  • 1.2.2 Defense mechanisms ............................................................................ 14
  • 1.3 Pathogens .................................................................................................... 15
  • 1.3.1 Infection strategies ............................................................................... 15
  • 1.3.2 EPEC/EHEC ........................................................................................ 16
  • 1.3.3 Salmonella ........................................................................................... 17
  • 1.3.4 Shigella ................................................................................................ 18
  • 1.3.5 T3SS ..................................................................................................... 19
  • 1.4 WxxxE ......................................................................................................... 21
  • 1.4.1 Map ...................................................................................................... 22
  • 1.4.2 EspT ..................................................................................................... 23
  • 1.4.3 SifA/B .................................................................................................. 24
  • 1.4.4 IpgB1/2 ................................................................................................ 24
  • 1.5 Aim .............................................................................................................. 25
  • 2 Results ................................................................................................................ 26
  • 2.1 Y2H screen .................................................................................................. 26
  • 2.2 Recombinant proteins and subsequent pull down assays ............................ 27
  • 2.2.1 EspT and its putative interactors .......................................................... 28
  • 2.2.2 SopB and its putative interactors ......................................................... 31
  • 2.2.3 IpgB1/2 and its putative interactors ..................................................... 32
  • 2.2.4 SifA/B and its putative interactors ....................................................... 35
  • 2.2.5 Map and its putative interactors ........................................................... 37
  • 2.2.6 Rhophilin CHMP4b interaction ........................................................... 42
  • 2.3 Co-immunoprecipitations ............................................................................ 43
  • 2.4 Microscopy .................................................................................................. 44
  • 2.4.1 Influence of WxxxE proteins on the actin cytoskeleton ...................... 44
  • 2.4.2 Map provokes a Rac1 like phenotype, RHPN1 induces mild stress fibers .................................................................................................... 48
  • 2.4.3 Map and RHPN1 can co-localize ......................................................... 51
  • 2.4.4 Co-transfection of small GTPases does not affect the RHPN1-Map interaction ............................................................................................ 53
  • 2.4.5 MitoTracker staining revealed co-localization between human RHPN1 and mitochondria ................................................................................. 54
  • 2.5 Infection assay ............................................................................................. 56
  • 2.6 Gentamycin protection assay ...................................................................... 60
  • 2.7 RHPN1 CHMP4b interaction ...................................................................... 64
  • 3 Discussion .......................................................................................................... 65
  • 3.1 EspT does not interact with Arf6 ................................................................ 66
  • 3.2 SopB does not interact with the V-ATPase subunit ATP6V1E1 ................ 67
  • 3.3 IpgB2 interacts with BBS4.......................................................................... 68
  • 3.4 The SifA – Rab9 interaction ....................................................................... 70
  • 3.5 The interaction between Map and human RHPN1 ..................................... 71
  • 3.6 Collaborative projects ................................................................................. 77
  • 3.7 Concluding remarks .................................................................................... 78
  • 4 Material .............................................................................................................. 80
  • 4.1 Chemicals .................................................................................................... 80
  • 4.2 Cell culture reagents and plasticware .......................................................... 80
  • 4.3 Bacterial strains ........................................................................................... 80
  • 4.4 Cell lines...................................................................................................... 81
  • 4.5 Plasmids ...................................................................................................... 81
  • 4.6 Oligonucleotides ......................................................................................... 85
  • 4.7 Antibodies ................................................................................................... 86
  • 4.8 Kits .............................................................................................................. 87
  • 5 Methods ............................................................................................................. 88
  • 5.1 Cultivation of bacteria ................................................................................. 88
  • 5.1.1 Preparation of RbCl competent bacteria .............................................. 88
  • 5.1.2 Transformation of chemical competent E.coli ..................................... 88
  • 5.2 Cell culture methods ................................................................................... 89
  • 5.2.1 Media ................................................................................................... 89
  • 5.2.2 Cultivation ........................................................................................... 89
  • 5.2.3 Freezing and thawing of cells .............................................................. 90
  • 5.2.4 Transfection of mammalian cells ......................................................... 90
  • 5.2.5 Cell preparation for fluorescence microscopy ..................................... 91
  • 5.2.6 Infection assay ..................................................................................... 91
  • 5.2.7 Gentamicin protection assay ................................................................ 91
  • 5.3 Molecular biological methods ..................................................................... 92
  • 5.3.1 Yeast two hybrid screen ....................................................................... 92
  • 5.3.2 Polymerase chain reaction (PCR) ........................................................ 92
  • 5.3.3 Inverse PCR ......................................................................................... 93
  • 5.3.4 Site directed mutagenesis ..................................................................... 93
  • 5.3.5 DNA gel electrophoresis and gel extraction ........................................ 93
  • 5.3.6 DNA restriction digest ......................................................................... 94
  • 5.3.7 Ligation of DNA fragments ................................................................. 94
  • 5.3.8 Gateway cloning .................................................................................. 94
  • 5.3.9 DNA amplification .............................................................................. 94
  • 5.3.10 DNA sequencing .................................................................................. 95
  • 5.4 Biochemical methods .................................................................................. 95
  • 5.4.1 Recombinant protein purification ........................................................ 95
  • 5.4.2 Pull down analysis ............................................................................... 97
  • 5.4.3 Co-immunoprecipitation ...................................................................... 98
  • 5.4.4 Protein gel electrophoresis (SDS-PAGE) ............................................ 98
  • 5.4.5 Coomassie staining .............................................................................. 99
  • 5.4.6 Western blotting and protein detection .............................................. 100
  • 5.4.7 Stripping of PVDF