Exploitation of host cellular pathways by Chlamydia trachomatis [Elektronische Ressource] / Sebastian Banhart. Gutachter: Thomas F. Meyer ; Thomas Sommer ; Andreas Herrmann

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2012
• Nombre de lectures: 46
• Langue: English
• Poids de l'ouvrage: 12 Mo

Extrait

Exploitation of host cellular pathways
by Chlamydia trachomatis


D i s s e r t a t i o n

zur Erlangung des akademischen Grades
d o c t o r r e r u m n a t u r a l i u m
(Dr. rer. nat.)
im Fach Biologie



eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Dipl.-Biol. Sebastian Banhart




Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Jan-Hendrik Olbertz

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann



Gutachter: 1. Prof. Dr. Thomas F. Meyer
2. Prof. Dr. Thomas Sommer
3. Prof. Dr. Andreas Herrmann

Tag der mündlichen Prüfung: 19.12.2011












































Todo começo é involuntário.
All beginnings are involuntary.

– Fernando Pessoa


Parts of this work have been or will be published under the following titles:

Banhart S.*, Mehlitz A.*, Mäurer A.P., Kaushansky A., Gordus A.G., Zielecki J., MacBeath G., Meyer
T.F. 2010. Tarp regulates early Chlamydia-induced host cell survival through interactions with the
human adaptor protein SHC1. J Cell Biol. 190(1):143-57. (* equal contribution)

Banhart S., Mehlitz A., Fuchs B., Schiller J., Heuer, D., Meyer, T.F. Global lipid analysis reveals
essential role of cardiolipin synthase 1 for Chlamydia replication. (in preparation)

Table of contents

Table of contents
Table of contents .................................................................................................................................... 1
Abstract ................................................................................................................................................... 4
Zusammenfassung .. 6
1 Introduction .................................................................................................................................... 9
1.1 Chlamydiae . 9
1.1.1 Taxonomy of Chlamydia ....... 9
1.1.2 Pathology of Chlamydia ...................................................................................................... 10
1.1.3 Developmental cycle of Chlamydia ..................................................................................... 11
1.2 Protein secretion systems ........ 13
1.2.1 Type III secretion system of Chlamydia .............................................................................. 13
1.2.2 The effector protein Tarp .................................... 14
1.3 Host-pathogen interplay .......... 14
1.3.1 Chlamydia and the MAPK pathway .................................................................................... 15
1.3.2 Chlamydia and apoptosis .................................................................................................... 16
1.3.3 Chlamydia and nutrient acquisition .................... 18
1.3.4 Chlamydia and lipids ........................................................................................................... 19
1.4 Aim of this study ...................... 20
2 Materials and methods ................. 22
2.1 Materials and general methods ............................................................................................... 22
2.1.1 Cell lines and bacteria ......................................... 22
2.1.2 Reagents, antibodies and constructs .................................................. 22
2.1.3 Infection time courses ........................................ 23
2.1.4 Statistical analysis ............................................... 23
2.2 DNA techniques........................................................................................ 23
2.2.1 Transfections ....................................................... 23
2.2.2 DNA microarrays and analysis ............................ 24
2.2.3 qRT-PCR analysis ................................................................................. 24
2.3 Protein techniques ................................................... 26
2.3.1 Peptide synthesis and protein microarrays ........................................ 26
2.3.2 Pull-down assays and immunoblotting ............... 28
2.4 Lipid techniques ....................................................................................................................... 28
2.4.1 Lipid extraction ................... 28

1 Table of contents

2.4.2 MALDI-TOF mass spectrometry .......................................................................................... 29
2.5 Microscopy ............................................................... 30
2.5.1 Immunofluorescence staining and microscopy .................................................................. 30
2.5.2 Live-cell fluorescence microscopy ...................................................... 30
2.6 Other methods ......................................................................................... 30
2.6.1 Invasion, inclusion formation, and progeny assays ............................ 30
2.6.2 Apoptosis induction and detection, data acquisition and analysis..................................... 31
2.7 Computational methods .......................................................................... 32
2.7.1 Multiple sequence alignment ............................. 32
2.7.2 Prediction of SH3 interactions ............................................................................................ 32
2.7.3 Modeling of Tarp signaling.................................. 32
2.7.4 Docking simulation .............................................................................. 33
3 Results and discussion .................................................. 35
3.1 Preface ..................................................................................................... 35
3.2 Part I: Interactions of host cell proteins with the effector protein Tarp ................................. 36
3.2.1 Peptide design of Tarp interaction sites ............................................. 36
3.2.2 Quantitative SH2/PTB interactome analysis of Tarp N-terminus ....... 38
3.2.3 Quantitative SH3 interactome analysis of Tarp C-terminus ............................................... 42
3.2.4 Pathway analysis of N-terminal interactome ..................................... 45
3.2.5 Pathway analysis of C-terminal interactome ...................................... 46
3.2.6 Validation of interactions between Tarp’s N-terminus and selected host cell proteins .... 47
3.2.7 Analysis of the interaction between Tarp’s C-terminus and NCK2 ..... 49
3.2.8 SHC1 activation and influence on MEK/ERK signaling during Chlamydia cell entry ........... 51
3.2.9 SHC1 activation and its transcriptional regulation during infection ................................... 56
3.2.10 SHC1 activation and its role as a survival stimulus ............................................................. 62
3.2.11 Discussion............................................................................................ 65
3.3 Part II: Changes in lipid composition induced by Chlamydia ................................................... 71
3.3.1 Analysis of lipids using MALDI-TOF mass spectrometry ..................... 71
3.3.2 Temporal changes in lipid composition during the chlamydial infection cycle .................. 75
3.3.3 Impact of cPLA2 and CRLS1 on chlamydial growth and progeny formation ...................... 83
3.3.4 Discussion............................................................................................................................ 86
4 Conclusions and outlook ............... 92
5 References .................................................................................................................................... 94

2 Table of contents

6 Appendix ..................................................................................................................................... 108
6.1 Supplemental material ........... 108
6.2 Abbreviations ......................................................................................................................... 131
6.3 List of figures .......................... 133
6.4 List of tables ........................... 135
6.5 Acknowledgments .................................................................................................................. 136
6.6 Publications ............................ 137
6.7 Selbständigkeitserklärung ...................................................................................................... 138

3 Abstract
Abstract
The human pathogen Chlamydia trachomatis has evolved to strongly rely on the host due to its
obligate intracellular replication and the acquisition of essential host cellular nutrients. This intimate
relationship requires the efficient exploitation of both signaling and trafficking pathways of the host
cell to ensure the pathogen’s differen