From pores to rafts to toll [Elektronische Ressource] : the adventures of L. monocytogenes on host cell membranes / von Ong'ondo Nelson Gekara

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Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

From Pores to Rafts to Toll-The
Adventures of L. monocytogenes on
Host Cell Membranes



Von Fachbereiches für Biowissenschaften und Psychologie,
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr.rer.nat.)


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


von Ong’ondo Nelson Gekara
aus Kisii, Kenya

Table of contents 2



































1. Referent: Professor Dr. Jürgen Wehland

2. Referent: Prof. Dr. Norbert Käufer

eingereicht am: 01.09.2005

mündliche Prüfung (Disputation) am:02.12.2005

Druckjahr: 2007



Table of contents 3
Vorveroffentlichungen der dissertation

Teilergebnisse aus dieser Arbeit wurden mit Genehmigung des Fachbereiches für
Biowissenschaften und Psychologie, vertreten durch den Mentor der Arbeit, in
folgenden Beiträgen vorab veröffentlicht:
Publikationen
Gekara, N.O. and Weiss, S. 2004. Lipid rafts clustering and signalling by
listeriolysin O. Biochem.Soc.Trans. 32:712-714

Gekara, N.O., Jacobs, T., Chakraborty, T., and Weiss, S. 2005. The
cholesterol-dependent cytolysin listeriolysin O aggregates rafts via
oligomerization. Cell Microbiol. 7:1345-1356.

Gekara, N.O., Westphal, K., Ma B., Groebe, L., and Weiss, S. The multiple
2+mechanisms of Ca signalling by listeriolysisn O (submitted)


Tagungsbeiträge
Gekara N.O., Jacobs, T, Ditmmar, KE, Chakraborty, T, and Weiss, S.
Activation of macrophages by Listeriolysin O involves aggregation of lipid rafts.
Immunobiology. 2003 208 (1-3):168 (34th Annual Meeting of the German
Society of Immunology and 3rd Meeting of European Mucosal Group, Berlin
Germany 2003).

Gekara, N.O., Jacobs,T., Chakraborty, T., and Weiss, S. Lipid rafts
aggregation and signalling by listeriolysin O. (Bioscience 2004 Conference,
Glasgow Scotland 2004).
Table of contents
1 Introduction ........................................................................................1
1.1 Target host cells of L. monocytogenes................................................................1
1.1.1 Macrophages and Dendritic cells (DCs) ...........................................................2
1.1.1.1 Kupffer cells .............................................................................................................. 2
1.1.1.2 Resident splenic macrophages................................................................................. 2
1.1.1.3 Infiltrating monocytes ................................................................................................ 3
1.1.1.4 Dendritic cells (DCs) ................................................................................................. 3
1.1.2 Granulocytes.....................................................................................................4
1.1.2.1 Neutrophils ................................................................................................................ 4
1.1.2.2 Mast cells .................................................................................................................. 4
1.1.3 Endothelial and Epithelial cells .........................................................................5
1.1.3.1 Enterocytes ............................................................................................................... 5
1.1.3.2 Hepatocytes 6
1.1.4 Lymphoid cells ..................................................................................................6
1.1.4.1 T cells........................................................................................................................ 6
1.2 Internalisation and intracellular life cycle of L. monocytogenes.......................7
1.3 Role of Listeriolysin in infection ...........................................................................9
1.3.1 What are the mechanisms of signal induction by LLO?..................................10
1.4 Induction of calcium signalling in host cells during interaction with bacterial
pathogens .........................................................................................................................11
1.5 Role of lipid rafts in the interaction of pathogens with hosts cells .................14
1.5.1 Conception of the raft hypothesis ...................................................................14
1.5.2 Physico –chemical properties of rafts .............................................................17
1.5.3 Criteria for association of proteins with rafts...................................................18
1.5.4 Caveolae.........................................................................................................18
1.5.5 Cellular and subcellular distribution of rafts.19
1.5.6 Lipid rafts in signal transduction .....................................................................20
1.5.7 Role of rafts / caveolae in infection.................................................................23
1.5.7.1 Lipid rafts in the internalisation of L. monocytogenes and other pathogens........... 24
1.5.7.2 Rafts as concentration platforms for toxins............................................................. 26
1.5.7.3 as signalling complexes for pathogens.......................................................... 26
1.6 Pathogen Recognition by Toll-like receptors ....................................................27
1.6.1 Signalling via TLRs .........................................................................................28
1.6.2 Role of TLRs in signal induction by L. monocytogenes ..................................30
1.7 Objectives of the work31
2 Materials and methods.....................................................................33
2.1 Animals..................................................................................................................33
2.2 Antibodies and reagents......................................................................................33
2.3 Cell lines and primary cells .................................................................................33 Table of contents II
2.4 Depletion of neutrophils and mast cells in mice ...............................................34
2+2.5 Ca flux measurements by flow cytometry .......................................................34
2.6 Measurement of β-Hexosaminidase activity ......................................................35
2.7 Infection of mast cells with L. monocytogenes.................................................35
2.8 TNF Bioassay........................................................................................................35
2.9 RT- PCR analysis..................................................................................................36
2.10 Neutralization of LLO with antibody and cholesterol........................................36
2.11 Preparation of Detergent Resistant Membranes (DRMs)..................................37
2.12 Separation of monomeric and oligomeric LLO..................................................37
2.13 Immunofluorescence staining and microscopy of J774 cells..........................37
2.14 Mast cells staining and microscopy ...................................................................39
2.15 Immunoprecipitation and western blot analysis ...............................................39
3 Results .............................................................................................41
3.1 Induction of calcium signals in host cells by LLO ............................................41
2+3.1.1 LLO induces release of Ca from intracellular stores in mast cells................41
3.1.2 LLO and L. monocytogenes induce de novo synthesis and secretion of
proinflammatory factors by mast cells ............................................................................43
3.1.3 The TNF- α gene transcriptional activation by LLO is pore dependent, occurs
2+via NFAT activation, and occurs independently of extracellular Ca .............................44
3.1.4 LLO induced ER injury causes release of ER components ..........................46
2+3.1.5 Prolonged LLO treatment leads to the depletion of intracellular Ca stores..47
3.2 Interaction of LLO with Lipid Rafts.....................................................................51
3.2.1 The role of cholesterol in the binding of LLO to plasma membranes .............51
3.2.2 LLO partitions into Detergent Resistant Membranes (DRMs).........................52
3.2.3 Effect of LLO on the membrane distribution of raft associated molecules......53
3.2.4 Mechanism of rafts aggregation by LLO.........................................................59
3.2.5 LLO induces tyrosine phosphorylation in a raft aggregation dependent manner
........................................................................................................................62
3.2.6 Activation of Lyn and Syk by LLO................................