From pores to rafts to toll [Elektronische Ressource] : the adventures of L. monocytogenes on host cell membranes / von Ong
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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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122 Pages
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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.

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Published 01 January 2007
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Exrait







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...................................................................64
3.3 Role of mast cells in the control of Listeriosis..................................................66
3.3.1 L. monocytogenes induces cytokine and chemokine transcription via LLO
dependent and independent mechanisms......................................................................67
3.3.2 L. monocytogenes induces recruitment of neutrophils into the peritoneum via
LLO dependent and independent mechanisms..............................................................69
3.3.3 Mast cells significantly contribute to the recruitment of neutrophils by LLO and
L. monocytogenes ..........................................................................................................71
Table of contents III
3.3.4 Accumulation of TNF- α in the peritoneal cavity of mice infected
intraperitoneally with L. monocytogenes ........................................................................71
3.3.5 Mast cell dependent recruitment of neutrophils is required for listerial
clearance ........................................................................................................................74
3.4 Role of Toll-like receptors (TLR) in activation of mast cells by L.
monocytogenes................................................................................................................77
3.4.1 Activation of NF-κB by L. monocytogenes......................................................77
3.4.2 Role of TLR signalling in the activation of proinflammatory cytokines
/chemokine genes ..........................................................................................................78
4 Discussion........................................................................................81
2+4.1 The Influx and Efflux of Ca induced by LLO in target cells- the consequence
of double membrane perforation ....................................................................................81
4.2 LLO induces signalling in host cells via the aggregation of lipid rafts...........84
4.3 Role of mast cells in the control of L. monocytogenes infection ....................88
4.4 Role of TLRs signalling in the activation of mast cells by L. monocytogenes...
................................................................................................................................90
5 Summary..........................................................................................92
6 References.......................................................................................94

1 Introduction
The Gram-positive bacteria Listeria monocytogenes is the food borne etiological
agent of Listeriosis whose manifestations include septicemia, meningitis (or
meningoencephalitis), encephalitis and abortions (1). Although Listeriosis targets
mainly immunocompromised populations such as pregnant women and fetus, the
elderly, diabetics, AIDS or cancer patients, healthy individuals can also develop the
disease, particularly if the foodstuff is heavily contaminated with highly virulent
variants of the pathogen (2). Although almost all Listeria strains that induce sepsis,
meningitis and encephalitis, as well as many other manifestations particularly in
immunocompromised individuals are susceptible to most of the common antibiotics,
the cure rate is only approximately 70% (2). Thus, if unrecognized and not treated in
time, Listeriosis is associated with rates of fatality as high as 70 %. In addition,
Listeriosis causes serious problems in livestock farming with 10% of animal death
and 50% abortion being due to Listeria infection (3). Thus understanding the
pathogenic mechanisms of L. monocytogenes is of high biomedical importance.
Since it is well characterized and one of the most easily manipulated bacterial
pathogens, L. monocytogenes is also a ´favourite` model pathogen for immunologists
and microbiologists commonly used for characterizing the mammalian immune
system and the pathogenic mechanisms of intracellular microorganisms.
1.1 Target host cells of L. monocytogenes
The natural route of infection with L. monocytogenes is through the gastrointestinal
tract. Upon ingestion, bacteria invade the intestinal epithelium and /or Peyer´s
patches, and disseminate via the bloodstream to the liver and spleen (1). Multiple
components of the immune system are involved in protection from Listeria infection.
Innate immunity controls pathogen replication during the first 2-3 days after infection.
Early host defenses either eliminate the microbe or modify the slope of the growth
curve, thereby preventing overwhelming sepsis and setting the stage for T-cell
dependent elimination of the pathogen (4). The significance of innate immunity to L.
monocytogenes is emphasized by the remarkable ability of mice deficient in both T-
cell and humoral immunity to control the early phase of infection (5;6). The main
target host cells involved in the survival as well as control of L. monocytogenes are
discussed below. 1. Introduction 2
1.1.1 Macrophages and Dendritic cells (DCs)
1.1.1.1 Kupffer cells
Upon intravenous inoculation, more than 60% of the Listeria inoculum is cleared from
the bloodstream by the liver within 10 min (7). This is due to the Kupffer cells. Kupffer
cells are resident tissue macrophages of the liver and account for 80-90% of the total
fixed tissue macrophages in the body (4). Kupffer cells are more dense in the
periportal region, and are optimally located for response to systemic bacteria and
bacterial products transported from the gut to the liver via the portal vein (8;9). Mice
deficient in Kupffer cells exhibit significant increases in blood Listeria and decreases
in liver Listeria 10 min after injection (4). Thus, one of the very early actions of
Kupffer cells is to physically trap the majority of Listeria in the liver during systemic
infections. Since more that 90% of liver bateria in the later stage of infection are
associated not with Kupffer cells but hepatocytes, (7;10), it has been suggested that
Kupffer cells clear bacteria via adherence rather than phogocytosis (4). In addition,
Kuppfer cells can also inhibit Listeria growth in by stander cells in vivo, either by
physical interaction or secretion of soluble factors, including interleukin (IL)-6, IL-12
IL-1 α, tumor necrosis factor alpha (TNF- α) and nitric oxide (NO). These mediators
are capable of promoting proinflammatory responses and antimicrobial activity of
other cell populations within the liver (4;11;12).
1.1.1.2 Resident splenic macrophages
Rapid removal of pathogens from the circulation by secondary lymphoid organs is
requisite for successful control of infection. Blood-borne antigens are trapped mainly
in the splenic marginal zone. Selective depletion of marginal zone macrophages and
marginal metallophilic macrophages caused impaired control of infection. Depletion
these cells did not however, limit Ag presentation since Listeria-specific unimpaired
protective T cell immunity was induced (13). Therefore, marginal zone macrophages
and marginal metallophilic macrophages are crucial for trapping of particulate Ag but
+dispensable for Ag presentation (13). Muraille et al (14) have shown that CD8 T cell
priming is indeed mediated by dendritic cells rather than macrophages. However,
since unpublished data by Jablonska et al show that Listeria infects and induces the
+expression of the CC-chemokine ligand (CCL2) in a ERT9 splenic macrophage 1. Introduction 3
subtype it seems possible that listerial clearence by splenic macrophages is
mediated via direct as well as indirect mechanisms.
1.1.1.3 Infiltrating monocytes
The recruitment of monocytes is a feature of the inflammatory response to infection
with Listeria and is essential for bacteria clearance. Circulating monocytes express
CCR2, the chemokine receptor for CCL2 which as mentioned above is induced in
macrophages by L. monocytogenes. Mice that lack CCR2 or CCL2 show a markedly
increased susceptibility to infection to L. monocytogenes. Such mice have
considerably lower levels of TNF-α and inducible nitric oxide oxygen synthase
(iNOS), factors that are essential for defence against L. monocytogenes infection
(15). The role of macrophages in bacterial clearance was also demonstrated by the
fact that when their recruitment to the sites of infection was blocked using a
monoclonal antibody against the complement receptor 3 (also known as CD11b-
CD18), the mice became more susceptible to Listeria infection (16).
1.1.1.4 Dendritic cells (DCs)
Although DCs play a major role in antigen presentation and generation of antilisterial
T-cell responses, several studies show that DC are not a significant in vivo reservoir
of L. monocytogenes (14;17). It is therefore proposed that the acquisition and
presentation of Listeria antigen occurs via cross-presentation, a process that involves
engulfment and degradation of infected cells (18). In addition to priming T cell
responses, which are required for sterile clearance of Listeria, some subsets of DCs
are also involved in the innate immune responses against Listeria. As mentioned
above, TNF-α and iNOS are essential for defense against Listeria infection. Analysis
of spleens from infected wild-type mice revealed a DC population that produces high
levels of TNF- α and iNOS and these cells are absent from CCR2 deficient mice.
These observation indicate that the newly discovered population of TNF- α and
iNOS-producing DCs (known as TipDCs) is involved in the control of Listeria growth
+ in vivo (19). It is worth to mentioning that CD8α lymphoid DCs have also been
implicated as an important innate source of IFN-β during Listeria infection (20).
1. Introduction 4
1.1.2 Granulocytes
1.1.2.1 Neutrophils
In mice, neutrophils represent approximately 15% of peripheral white blood cells.
During bacterial infection, neutrophils efflux from the bone marrow into the peripheral
blood, then into the sites of infection. Once localized at the site of infection,
neutrophils may contribute to antibacterial resistance by killing bacteria, lysing
infected host cells, stimulating apoptosis in infected cells, and / or secreting cytokines
that suppress replication of intracellular bacteria within infected cells.
That neutrophils play a part in resistance to Listeria infections is demonstrated by the
fact that neutrophil-deficient mice, as well as mice unable to mobilize neutrophils
upon infection, exhibit dramatic increases of Listeria in the liver and spleen (see
results section and (21;22).
1.1.2.2 Mast cells
Mast cells are derivatives of hematopoietic progenitor cells that migrate into virtually
all vascularized tissues, where they complete their maturation (23). Mature mast cells
normally reside close to epithelia, blood vessels, nerves, near smooth muscle cells,
mucus-producing glands, in the airways and in the gastrointestinal tract (23). Under
certain circumstances, morphologically identifiable mast cells can migrate locally
within tissues, including into epithelia (24;25). In some species, including murine
rodents, mast cells also occur within mesothelium-lined cavities, such as the
peritoneal cavity (23;24).
Mast cells are known mainly for their involvement in mediating various harmful
inflammatory reactions in the host, the best known of these being immunoglobulin E
(IgE)-mediated immediate-type hypersensitivity reactions. Other pathologic
processes involving mast cells include inflammatory bowel disease, autoimmune
diseases, tissue remodeling and fibrosis (26). The contribution of mast cells to these
chronic inflammatory conditions is exacerbated by the fact that mast cells are
extremely long lived, with a lifespan of months to years, and because of their capacity
to proliferate at sites of inflammation (27).
Although the focus on mast cells has largely been in relation to allergic or
autoimmune diseases, their role in mediating bacterial clearance at sites of infection
is now well recognized (28). After activation, mast cells exert their biological effects