Host cell death modulation by Chlamydia trachomatis [Elektronische Ressource] / Manu Sharma. Gutachter: Thomas Rudel ; Thomas Meyer ; Thomas Adam

Manu Sharma

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 58
• Langue: English
• Poids de l'ouvrage: 10 Mo

Extrait

Host cell death modulation by Chlamydia 
trachomatis 
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Manu Sharma
geboren am 11.11.1980 in New Delhi
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Lutz-Helmut Schön
Gutachter: 1. Prof.Thomas Rudel
2. Prof Thomas Meyer
3. Prof.Thomas Adam
Tag der mündlichen Prüfung: 20 July, 2009 Table of Contents
ZUSAMMENFASSUNG................................................................................................................................. 5
SUMMARY....................... 7
ABBREVIATIONS.......... 9
1 INTRODUCTION.................................................................................................................... 12
1.1 CHLAMYDIA... 12
1.1.1 Chlamydial taxonomy.............................................................................................................. 12
1.1.2 Chlamydial diseases, medical significance ............................................................................. 13
1.1.3 Life cycle of Chlamydia ........................................................................................................... 14
1.2 APOPTOSIS .................................................................................................................................... 15
1.2.1 Caspases... 16
1.2.2 The Inhibitor of Apoptosis Proteins......................................................................................... 18
1.2.3 Pathways of Apoptosis............................................................................................................. 19
1.2.4 Bcl-2 family of proteins 22
1.2.5 Pro-survival Bcl-2 proteins ..................................................................................................... 25
1.3 MYELOID CELL LEUKAEMIA-1 ...................................................................................................... 25
1.4 HYPOXIA INDUCIBLE FACTOR-1.................................................................................................... 26
1.4.1 HIF-1  and anti-apoptosis 27
1.4.1 HIF-1  stabilization during Chlamydia pneumoniae infection............................................... 27
1.5 APOPTOSIS IN DEFENCE AGAINST INFECTIONS............................................................................... 28
1.6 CHLAMYDIA AND APOPTOSIS ........................................................................................................ 28
2 RESULTS ................................................................................................................................. 32
2.1 CHARACTERIZATION OF APOPTOSIS RESISTANCE IN CELLS INFECTED WITH CHLAMYDIA
TRACHOMATIS ............................................................................................................................ 32
2.1.1 C. trachomatis inhibits apoptosis induced by different stimuli................................................ 32
2.1.2 Activation of caspase-3 was inhibited in Chlamydia-infected cells......................................... 33
2.1.3 Caspase-8 was processed in Chlamydia infected cells upon apoptosis induction................... 34
2.2 THE INHIBITOR OF APOPTOSIS PROTEIN – COMPLEXES ARE REQUIRED FOR APOPTOSIS RESISTANCE
IN CHLAMYDIA TRACHOMATIS INFECTED CELLS......................................................................... 35
2.2.1 cIAP-2 expression is up-regulated during an infection with C. trachomatis........................... 36
2.2.2 IAPs are required for apoptosis resistance in the infected cells.............................................. 37
2.2.3 IAPs interact with each other, existing in large heteromeric native complexes ...................... 38
2.2.4 The formation of IAP complexes is not cell type specific ........................................................ 39
2.2.5 Smac and caspase-3 get recruited to the IAP-IAP complex during apoptosis......................... 40
2.3 BH3 ONLY PROTEINS ARE NOT DEGRADED IN CELLS INFECTED WITH CHLAMYDIA TRACHOMATIS .... 43
2.4 RNA INTERFERENCE SCREEN TO IDENTIFY FACTORS INVOLVED IN INFECTION INDUCED APOPTOSIS
RESISTANCE ................................................................................................................................46


2.4.1 Pathway analysis of the targets from the screen implicates HIF-1  as a key regulator of
apoptosis resistance ................................................................................................................ 47
2.5 MCL-1 CONSTITUTES THE PROMINENT BLOCK UPSTREAM OF THE MITOCHONDRIA, TO PREVENT
APOPTOSIS IN CHLAMYDIA INFECTED CELLS............................................................................... 49
2.5.1 Mcl-1 gets up regulated during chlamydial infection.............................................................. 49
2.5.2 Mcl-1 is up regulated in a MAPK dependant process ............................................................. 49
2.5.3 Mcl-1 is required for resistance to apoptosis in the infected cells .......................................... 51
2.5.4 The inhibition of PI3 Kinase sensitized infected cells to Staurosporine induced apoptosis. ... 52
2.5.5 Mcl-1 is required for the block in mitochondrial outer membrane permeabilization in the
infected cells, upon apoptosis induction.................................................................................. 53
2.5.6 Loss of Mcl-1 can rescue release of cytochrome c in the infected cells upon apoptosis
induction.................................................................................................................................. 56
2.5.7 Loss of Mcl-1 leads to caspase-9 activation in the infected cells, upon apoptosis induction .. 62
2.6 HIF-1 GETS STABILIZED AND MODULATES THE EXPRESSION OF ANTI-APOPTOTIC PROTEINS DURING C.
TRACHOMATIS INFECTION ........................................................................................................... 63
2.6.1 HIF-1  gets stabilized at the protein level during early infection, but gets degraded during
late time points of infection ..................................................................................................... 63
2.6.2 HIF1-  is not transcriptionally regulated during infection .................................................... 64
2.6.3 HIF-1  gets translocated to the nucleus during infection...................................................... 65
2.6.4 Hypoxia Inducible Factor is required for the transcriptional up-regulation of Mcl-1 after
infection with C.trachomatis ................................................................................................... 66
2.6.5 Loss of HIF-1  sensitizes Chlamydia infected cells to apoptosis............................................ 67
3 DISCUSSION ........................................................................................................................... 72
3.1 REQUIREMENT OF BACTERIAL AND HOST CELL PROTEIN SYNTHESIS FOR APOPTOSIS INHIBITION .. 73
3.2 BLOCK OF MITOCHONDRIAL OUTER MEMBRANE PERMEABILIZATION DURING INFECTION ............. 74
3.2.1 BH3 only proteins are not degraded during Chlamydia trachomatis infection....................... 74
3.2.2 Up-regulation of Mcl-1 is responsible for inhibiting mitochondrial outer membrane
permeabilization during C. trachomatis infection................................................................... 75
3.3 BLOCK IN CASPASE-3 ACTIVATION DURING INFECTION ................................................................. 76
3.3.1 IAPs exist in the form of native heteromeric complexes .......................................................... 77
3.4 MODULATION OF THE TRANSCRIPTION FACTOR HIF-1 TO REGULATE SURVIVAL GENES................ 79
3.5 DIFFERENT MECHANISM OF APOPTOSIS INHIBITION AT LATER TIME POINTS................................... 81
3.6 OUTLOOK...................................................................................................................................... 82
4 MATERIALS AND METHODS ............................................................................................ 84
4.1 MATERIALS.... 84
4.1.1 Cell lines... 84
4.1.2 Cell culture media ................................................................................................................... 84
3


4.1.3 Chemical reagents ................................................................................................................... 85
4.1.4 Primary antibodies .................................................................................................................. 85
4.1.5 Seconday antibodies ................................................................................................................ 86
4.2 BUFFERS ....................................................................................................................................... 87
4.2.1 Cell culture media for Chlamydia culture and maintenance................................................... 87
4.2.2 Indirect Immunofluoresce