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PEPTIDOGLYCAN RECOGNITION PROTEINS:
MAJOR REGULATORS OF DROSOPHILA IMMUNITY
PETER MELLROTH
DEPARTMENT OF GENETICS, MICROBIOLOGY AND TOXICOLOGY
STOCKHOLM UNIVERSITY
2005
Doctoral Thesis 2005
Department of Genetics, Microbiology and Toxicology,
Stockholm University, 106 91 Stockholm, Sweden
Abstract
All eukaryotic organisms have an innate immune system characterized by germ-line encoded
receptors and effector molecules, which mediate detection and clearance of microbes such as
bacteria, fungi, and parasites. Vertebrate animals have, in addition to innate immune
responses, evolved an adaptive immune system characterized by antibodies and T-cell
receptors. Insects in general and the fruit fly in particular, have in recent years become an
attractive model for the study of innate immunity. This is partly because it shares a great deal
of similarity with the mammalian innate immune system. The versatility of Drosophila as a
genetically tractable organism with a multitude of molecular-biology techniques available
makes it an excellent tool for studying innate immunity.
This thesis concerns the peptidoglycan recognition protein (PGRP) gene family in
the fruit fly. The family consists of thirteen genes, of which a few have been reported to be
part of the signaling pathways that regulates immune gene expression. PGRP proteins have
affinity for peptidoglycan, an essential bacterial cell wall molecule. The main goal of this
thesis has been to investigate which PGRP variants are required for immune pathway
induction and which ligands are being recognized.
Data presented show that the putative receptors have affinity for peptidoglycan, but
not for lipopolysaccharide, or the fungal cell wall polymer beta-glucan. PGRP-SA, receptor of
the Toll pathway, has a preference for peptidoglycan with a lysine in the crosslinking peptide.
Furthermore PGRP-LCx, receptor of the Imd pathway, shows strong affinity for all types of
peptidoglycan, both polymeric and monomeric. Activation of the Imd-pathway by polymeric,
DAP-type, peptidoglycan is dependent on PGRP-LCx, whereas activation by monomeric
peptidoglycan is dependent on both PGRP-LCx and PGRP-LCa. This result suggests that
receptor dimerization is required for activation. Indeed, monomeric peptidoglycan can induce
dimerization of the extracellular parts of PGRP-LCx and PGRP-LCa. However, PGRP-LCa
does not have peptidoglycan affinity but is apparently only involved as an adapter molecule.
These findings provide an explanation of the activation data for which a model is presented.
In a search for novel PGRP receptors I found two PGRP proteins that instead
displayed enzymatic activity towards peptidoglycan. They are of the N-actylmuramoyl Lalanine amidase type, which degrades peptidoglycan by splitting the crosslinking peptides
from the glycan strands. The PGRP-SC1B-degraded Staphylococcus aureus peptidoglycan
looses its immune elicitor capacity. This is in contrast to lysozyme-degraded peptidoglycan,
which is still immune stimulating. Amino acid sequence homology showed as many as six of
the thirteen Drosophila PGRPs to be potential enzymes. PGRP-SB1 is the other enzymatic
PGRP described within this thesis. It has a more narrow activity spectrum as compared to
SC1B implying that the fine-structure of peptidoglycan is important for the activity. In
addition, PGRP-SB1 has antibacterial activity against Bacillus megaterium.
In conclusion, receptor PGRP proteins binds bacterial peptidoglycan and triggers
immune gene pathways and enzymatic PGRPs have the capacity to reduce the elicitor
property of peptidoglycan.
© 2005 Peter Mellroth
ISBN 91-7155-105-0 (pp 1-58)
Intellecta docusys AB, Stockholm 2005
The scientific theory I like best is that the rings of Saturn
are composed entirely of lost airline luggage
Mark Russell
Table of contents
Abstract .................................................................................................................................... II
List of papers ........................................................................................................................ VII
The need of an immune system ............................................................................................... 8
The insect immune system..................................................................................................... 10
Barrier epithelia............................................................................................................................................... 10
Blood ................................................................................................................................................................ 10
Humoral responses ...................................................................................................................................... 11
Cellular responses........................................................................................................................................ 13
Immune organs................................................................................................................................................. 14
Signaling pathways................................................................................................................. 14
The Toll pathway.............................................................................................................................................. 15
The Imd pathway .............................................................................................................................................. 16
Toll and Imd are the major immune pathways ................................................................................................. 17
Other pathways ................................................................................................................................................ 18
Pattern recognition................................................................................................................. 18
Microbial immune targets ..................................................................................................... 19
Teichoic acids................................................................................................................................................... 19
Lipopolysaccharide .......................................................................................................................................... 20
Fungal β–1,3-glucan ........................................................................................................................................ 20
Peptidoglycan................................................................................................................................................... 21
Peptidoglycan synthesis and turnover ......................................................................................................... 23
Peptidoglycan hydrolyzing enzymes ........................................................................................................... 27
Recognition molecules............................................................................................................ 28
C-type Lectins................................................................................................................................................... 28
Toll-like receptors ............................................................................................................................................ 29
Nod receptors ................................................................................................................................................... 29
Gram-negative binding proteins ...................................................................................................................... 30
Peptidoglycan recognition proteins ................................................................................................................. 30
PGRP in Drosophila: Expression pattern and protein topology predictions ............................................... 31
Functional studies of Drosophila PGRPs .................................................................................................... 32
PGRPs in other insects ................................................................................................................................ 33
Mammalian PGRPs ..................................................................................................................................... 34
PGRP structures........................................................................................................................................... 35
The present study ................................................................................................................... 38
Aim........................................................................................................................................... 38
A PGRP enzyme (Paper I) ................................................................................................................................ 38
Enzyme characterization (Paper I) ................................................................................................................... 39
A peptidoglycan scavenger (Paper I)................................................................................................................ 40
A bactericidal PGRP amidase (Paper II) ......................................................................................................... 41
LPS – to be, or not to be, an elicitor of the Drosophila immune system (Paper III and IV) ............................. 41
PGRP-LC – three of a kind (Paper III and IV)................................................................................................. 42
Polymeric peptidoglycan recognition (Paper III, IV, and V) ........................................................................... 43
Monomeric peptidoglycan recognition (Paper III, IV, and V) ......................................................................... 44
Concluding remarks............................................................................................................... 45
Acknowledgements................................................................................................................. 47
References ............................................................................................................................... 49
List of papers
This thesis is based on the following publications:
I
Mellroth P., Karlsson J., Steiner H. (2003)
A scavenger function for a Drosophila peptidoglycan recognition
protein
J. Biol. Chem. 278:7059-64
II
Mellroth P., Steiner H. (2005)
PGRP-SB1 – an N-acetylmuramoyl L-alanine amidase with
antibacterial activity
Manuscript
III
Werner T., Borge-Renberg K., Mellroth P., Steiner H., Hultmark D.
(2003)
Functional diversity of the Drosophila PGRP-LC gene cluster in the
response to lipopolysaccharide and peptidoglycan
J. Biol. Chem. 278:26319-22
IV
Kaneko T., Goldman W.E., Mellroth P., Steiner H., Fukase K.,
Kusumoto S., Harley W., Fox A., Golenbock D., Silverman N.
(2004)
Monomeric and polymeric Gram-negative peptidoglycan but not
purified LPS stimulate the Drosophila IMD pathway
Immunity 20:637-49
V
Mellroth P., Karlsson J., Håkansson J., Schultz N., Goldman W.E.,
Steiner H. (2005)
Ligand-induced dimerization of Drosophila peptidoglycan
recognition proteins in vitro
Proc. Natl. Acad. Sci. USA 102:6455-60
The need of an immune system
In the evolutionary race, all organisms have evolved means to defend the
integrity of their genetic information contained in their genomes, which are
under constant attack from viruses and other selfish genetic elements with a
potential to modify or take control over the host’s DNA. Being sources of high
energy molecules and nutrients, all eukaryotic organisms must also defend their
cells and tissues from more faster propagating microorganisms. The enormous
biodiversity among bacteria, however, set high demands on the host immune
system in terms of detection and elimination. It must be specific enough to
distinguish bacteria from the own cells, or the multitude of molecules produced
by the own tissues. It must also be precise enough to kill the intruders without
causing too much damage to the own tissues.
The need of an efficient immune defense must certainly have been a
cornerstone in the childhood of eukaryotic evolution and molecules with
capacity to sense the presence of infectious microbes have been positively
selected. A battery of molecules with capacity to eradicate all kinds of bacteria
and fungi has also been contained in the evolutionary backpacks of the ancestors
to the higher organisms that exist today.
In common for the pre-existing eukaryotic life is the presence of an
innate immune defense with a typical architecture. It is built on germ-line
encoded sensors and effector molecules that recognizes and eliminates microbes
that enter the host tissues. It is aiming at structural features of the microbial cell
that has been seemingly unchanged during evolution. Even if distantly related
organisms are using different molecules for detection of microbes the target
epitopes are often the same. The recognition of a microbe is often linked to a
signaling pathway that will direct the expression of genes that either encode
effector molecules with capacity to kill the intruder, or encode cytokines and
chemokines that engage other cell types in the microbial elimination process
(Kimbrell and Beutler, 2001).
It is striking that many factors of the innate immune system indeed are
well conserved during evolution. A considerable number of genes encoding
recognition molecules, as well as effector molecules, and components of
signaling pathways, are conserved from organisms as divergent as humans and
flies. These genes must thus have been present in the common ancestor to
insects and mammals some 600 million years ago.
In vertebrate animals, an adaptive immune system has evolved in
addition to the innate immune defense. This adaptive immune system depends
on B- and T-cells of lymphatic origin, which has the capacity to form antibodies
and T-cell receptors through genetic rearrangement. In this elaborate process an
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enormous potential of molecular recognition is accomplished allowing for
detection of almost any possible microbial epitope. A major drawback of the
adaptive immune system is that it can take up to three weeks until a full
response has been established. However, once an intruder has been defeated,
memory cells are formed which ensure a rapid response in case of repeated
infections. The innate and adaptive systems works in parallel and the adaptive
system partly depends on signals in form of cytokines and co-factors produced
by cells of the innate system (Pasare and Medzhitov, 2004).
A common theme for most organisms is that their immune responses
are set in a resting-, or off-state, and is selectively activated whenever an
infectious agent manages to pass an epithelium barrier. There are several reasons
for this organization. One reason is that it is energetically costly to have a
system that is always in the on-state and constantly producing immune effector
molecules. Another reason is that most immune reactions are causing damages
also to own tissues. In mammals, for example, an over-activated immune system
can lead to a toxic-shock syndrome, often with fatal consequences. A third
reason is that microbes can more easily evolve immune circumventing
mechanisms if they are constantly being exposed to immune effector molecules.
Such resting immune systems are dependent on efficient sensors of microbes
that can selectively trigger the onset of the effector mechanisms. The microbial
recognition event is thus of central importance.
Bacteria are present everywhere in our environment. They have
occupied all ecological niches and are specialists in extracting nutrients and
energy from almost all imaginable sources. All high energetic biomasses like
soil or dead animal corpses, lacking an active immune system, are soon
overloaded with a multitude of decomposing bacteria. In our gut we have allied
bacteria that help us to break down the food we ingest. They are adapted to this
environment and also help us to exclude pathogens that first have to compete for
space and nutrients with these commensal bacterial communities. However,
these communities are so numerous that they by the tenfold exceed the number
of cells in our own bodies and it is therefore essential to control these bacteria
and limit their presence to the gut lumen. Also epidermal surfaces like our skin
harbor complex bacterial societies that are mostly not harmful to us but have a
potential to cause infection if they are getting access to a wound (Sansonetti,
2004).
The physiological barriers that constitute the topological border
dividing the organism’s interior from the exterior are guarded by the innate
immune system. If bacteria manage to pass such a barrier, an elaborate alarm
system is turned on triggering a multitude of responses aimed to track down and
eliminate the intruders. The responses can be of several different types. Premade protein cascades can be triggered, specialized cells release the content of
intracellular vesicles containing antibacterial factors, cells with a capacity to
engulf bacteria are activated, and a synthesis of antimicrobial peptides can be
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initiated. In mammals, small infections are often restricted to local and
temporary inflammations but in insects a more systemic response is mounted.
The bacterial detection event is critical to enable the innate immune system to
mount a response. It is therefore astonishing that it relies only on some handful
of receptors, but with a capacity to recognize almost all existing bacteria.
The insect immune system
Insects form the most successful (and sometimes irritating) animal group in
terms of species diversity and abundance. To date about one million species are
described, which is more than all other animal species combined, and they have
occupied most terrestrial and aerial ecological niches on our planet. Part of their
success is likely because they have evolved such an efficient immune defense.
Many insects thrive in nutrient-rich environments such as soil or excrements
with high contents of microbes and the need to efficiently recognize and battle
bacteria is obvious. I will, as follows, briefly highlight the most important
components of the fly immune system.
Barrier epithelia
The barrier epithelia serve as physiological shields against the environment and
prevent microbes from entering interior tissues. The insect cuticle is a
multilayered epithelium intertwined with protein matrixes and microfibers of
chitin. It can be soft and flexible as in larvae or hard and sclerotized as in most
adult insects. The digestive tract, tracheal tubules, and the genitals are also lined
with epithelial tissues composed of cell layers and chitin/ protein matrixes,
which secret factors like mucus and saliva. Cells of the midgut epithelium, for
example, produces an extracellular matrix called the peritrophic membrane
consisting of proteins, chitin, and proteoglycans and is working as a tough
protective filter against microbes (Lehane, 1997). Certain epithelial tissues are
also constitutively secreting antimicrobial peptides that aids in the protection
(Ryu et al., 2004).
Blood
Bacteria that are getting access to the body cavities through wounds in the
cuticle or by expressing virulence factors that enable passage through an
epithelial barrier will reach the insect blood, named the hemolymph. The
hemolymph is the main arena for the immune system and several different
responses are involved in bacterial clearance. There are both cellular and
humoral factors involved in the responses.
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Insects have an open blood system with a heart that pumps the
hemolymph to the body cavities surrounding the organs. The hemolymph
consists of 10% hemocytes (blood cells) and 90% serum. The total cell
concentration of the hemolymph is considerably lower than in human blood due
to the lack of erythrocytes. In the absence of red blood cells, the appearance of
the hemolymph ranges from clear to yellowish or greenish depending on insect
species.
Humoral responses
The phenoloxidase system
A melanization reaction can be triggered by the prophenol oxidase (PPO)
cascade (Cerenius and Soderhall, 2004). Anyone who has injured a large insect
has seen that the blood coming out is soon changing from clear to black. This
blackening is due to the formation of the dark pigment melanin, which is the end
product of the PPO cascade. The melanization reaction is considered to be an
immune defense reaction even if the melanin itself is not especially harmful to
microbes. However, some of the intermediates of the reaction are radicals and
reactive oxygen species that might be harmful to bacteria. The reaction is
catalyzed by the active phenoloxidase (PO) that catalyses oxidation of tyrosine
to orthoquinones, which spontaneously polymerize to melanin. The
enzymatically inactive PPO is cleaved into active PO in a process tightly
controlled by serine proteases and their inhibitors. In many insects a pattern
recognition receptor activates the cascade upon microbial infection (Ma and
Kanost, 2000; Ochiai and Ashida, 1999; Yoshida et al., 1996; Yu et al., 1999)
but in Drosophila no such receptor has been directly linked to PPO activation.
However, it is shown that PPO activation is in part dependent on the Toll
pathway (De Gregorio et al., 2002a; Ligoxygakis et al., 2002b) and that
overexpression of PGRP-LE can induce melanization in larvae (Takehana et al.,
2002). Melanin is deposited on parasites and becomes part of capsules and
noodles formed around parasites by blood cells (see below). The
prophenoloxidase cascade is present in insect and crustacean hemolymph but is
not found in any deuterostome organism.
Clotting
The clotting system is another humoral system that can rapidly be activated if
the cuticle is wounded (Bidla et al., 2005). Especially in larvae with its soft
cuticle it is vital to stop the bleeding because the blood pressure makes them
prone to leak. The molecular mechanism of how clot formation is triggered is
not clear but it is evident that a soft clot is soon formed after wounding (Scherfer
et al., 2004). This clot contains various proteins, including lectins but also
ruptured blood cells. The soft clot will eventually be further cross-linked and
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made hard by the melanization reaction. The clot will not only stop the blood
from leaking out, it will also trap and hinder bacteria from entering the body
cavities.
Inducible antimicrobial peptides
Even if the capacity of the insect to fight bacterial infections had been
previously studied by scientists like Louis Pasteur and Elie Metchnikoff, the
first biochemical analysis of anti-microbial factors in insect hemolymph was
initiated by Hans G. Boman and colleagues in the late 1970s (Boman and
Steiner, 1981). They discovered and characterized the first anti-microbial
peptides and this became a starting point for a new field that was aiming to map
the components of the insect immune system.
The first anti-microbial peptide described was isolated from the
Lepidoptera species Hyalophora cecropia and was named cecropin (Steiner et
al., 1981). It was shown to be a cationic peptide of 37 amino acid residues
having an unordered structure in solution. The net positive charge confers
affinity for negatively charged bacterial surfaces. When cecropin binds to the
bacterial membrane it folds into an ordered conformation consisting of two
helices one of which being amphipathic (Steiner, 1982). Cecropin induces
bacterial lysis because the peptide interferes with the integrity of bacterial
membranes, presumably by forming leaky pores or patches. Cecropin has a
broad spectrum of activity and potently kills both Gram-negative and Grampositive bacteria and is also active against fungi. In Drosophila, a total of seven
different anti-microbial peptides plus isoforms are described (reviewed in (Imler
and Bulet, 2005)).
Attacin, a glycine-rich protein of 20 kDa is mainly active against
Gram-negative bacteria and interferes with both bacterial membrane integrity as
well as outer membrane protein gene expression (Carlsson et al., 1991).
Drosocin and metchnikowin are proline-rich peptides with rather different
antimicrobial spectra. Drosocin is a glycosylated peptide, which is active against
Gram-negative bacteria whereas metchnikowin is active against Gram-positive
bacteria and fungi (Bulet et al., 1993; Levashina et al., 1995). Diptericin is a 9
kDa peptide with a short proline-rich N-terminal domain and a glycine-rich Cterminal domain and is active against some Gram-negative bacteria (Wicker et
al., 1990). Drosomycin and defensin are cysteine-rich peptides that form cyclic
domains through disulfide bridge formation (Dimarcq et al., 1994; Fehlbaum et
al., 1994). Drosomycin, an 8 kDa peptide containing four S-S bridges, has
potent antifungal activity. In addition to these inducible peptides Drosophila
also expresses seven lysozyme genes (Daffre et al., 1994). They are all of the
classical lysozyme type cleaving the glycan strands of peptidoglycan by
hydrolyzing the bond between N-acetyl-muramic acid (MurNAc) and N-acetylglucoseamine (GlcNAc) residues.
12
The bactericidal potency of the anti-microbial peptides was clearly
illustrated in a study on Drosophila flies with deficient immune pathways. These
flies are sensitive to bacterial infections but when manipulated to express a
single anti-microbial peptide, an almost wild type phenotype was obtained
(Tzou et al., 2002).
Upon infection, a massive anti-microbial peptide expression is
induced in the fatbody and the peptides are exported to the hemolymph. The
expression is regulated by two signaling pathways, the Toll and the Imd
pathway (reviewed in (Hultmark, 2003; Royet et al., 2005)). The Toll pathway
mainly regulates the antifungal response, e.g. drosomycin expression, while the
Imd pathway regulates the anti-bacterial peptide genes, e.g. diptericin and
attacin. Even if the fatbody is the main site of expression, also blood cells are
expressing antimicrobial peptides and contribute to the systemic humoral
response (Johansson et al., 2005). In addition to the systemic response, barrier
epithelial cells are capable of mounting a local inflammatory response involving
antimicrobial peptide expression (Onfelt Tingvall et al., 2001; Tzou et al., 2000).
In these tissues the immune gene induction is solely dependent on the Imd
pathway.
Some epithelial tissues also express anti-microbial peptides
constitutively. The Drosophila lysozyme genes, for example, have a constitutive
expression restricted to different parts of the gut (Daffre et al., 1994). This fact
possibly reflects the lifestyle of the fly, which is often feeding on decaying
fruits.
Cellular responses
Blood cells
In Drosophila there are three classes of hemocytes described; plasmatocytes,
lamellocytes, and crystal cells. The plasmatocytes is by far the most abundant
cell type (Lanot et al., 2001). They are multi-functional cells that mostly
resemble mammalian monocyte/ macrophage lineages. Being professional
phagocytes, they are capable of efficiently engulf invading bacteria as well as
own apoptotic cells. These are receptor-mediated responses and, at least partly,
dependent on the expression of the PGRP-LC and croquemort receptors,
respectively (Franc et al., 1996; Ramet et al., 2002b). Plasmatocytes are also
involved in other immune-related responses such as synthesizing inducible antimicrobial peptides and producing important clotting factors. Recently they were
also reported to be the primary producers of the endogenous peptide-cytokine
spätzle (Irving et al., 2005). The crystal cells constitute about 5% of the blood
cell population in larvae but are apparently absent in adult flies. These cells are
characterized by having crystalline inclusion bodies of the prophenoloxidase
enzyme required for the melanization reaction. Crystal cells are either
circulating or sessile, sitting together with plasmatocytes in contact with the
13
cuticular epithelial layer (Lanot et al., 2001). If the epidermis gets wounded, the
crystal cells will rupture and release their content to the surrounding tissues.
This ensures a local melanization reaction at the wound-site and thus prevents
the cytotoxic radicals produced to be formed throughout the hemocel.
Lamellocytes are not present in healthy individuals but can form in lymph
glands when larvae are under attack by parasites (Lanot et al., 2001). These cells
are flat-shaped and are mainly involved in encapsulation of foreign objects that
are too large to phagocytose. This specialized cell type has possibly evolved as a
defense response against parasitic hymenoptera species that are specialized in
laying their eggs in insect larvae. Lamellocytes moves towards and surround
parasite eggs to form tough capsules together with melanin.
Immune organs
The fatbody is the major immune-related organ of insects. It is a functional
equivalent of the mammalian liver and is the main site for synthesis of humoral
factors taking part in the systemic immune response. Upon infection, a massive
expression of immune genes is induced in the fatbody. Anti-microbial peptides
are rapidly secreted to the hemolymph and can reach as high concentrations as
100 µM (Meister et al., 2000; Samakovlis et al., 1990).
In the lymph glands, hematopoiesis (blood cell formation) takes
place during larval stages, plasmatocytes proliferate, and lamellocytes
differentiate under special conditions (Lanot et al., 2001).
Signaling pathways
The unraveling of how the immune-gene expression is controlled has been one
of the main interests in the field during the last decennia. An important clue to
how the gene-expression was regulated came when researchers examined the
promoter regions for some anti-microbial peptide genes and found that they
contained regulatory κB motifs (Engstrom et al., 1993; Sun and Faye, 1992).
These motifs were known to be present in promoter regions in mammalian genes
involved in inflammation responses controlled by nuclear factor κB (NFκB).
This transcriptional activator is found in the cytoplasm bound to an inhibitory
protein, IκB. When the signaling cascade is activated, the IκB protein is
phosphorylated and degraded, leading to the unmasking of a nuclear import
signal on NFκB. This promotes a translocation of NFκB from the cytoplasm to
the nucleus where it binds to κB motifs and activates transcription. In
Drosophila, three different Rel-homology proteins (homologous to NFκB) were
found to interact with κB motifs and one of those, dorsal, had been shown to be
an activator of the Toll-pathway, which defines the dorso-ventral polarity in
Drosophila embryogenesis (Ip et al., 1991; Thisse et al., 1991). It was soon
14
evident that the same signaling pathway was utilized by the immune system in
larval and adult stages but instead with another transactivator, named dorsalrelated immunity factor (Dif) (Ip et al., 1991; Ip et al., 1993; Lemaitre et al.,
1996; Meng et al., 1999; Rosetto et al., 1995). Interestingly, most of the
components in the Toll pathway are homologous to the interleukin-1 receptor
pathway in mammals. These findings led to the important discovery of the Tolllike receptors (TLRs), which are now known to be cornerstones for microbial
recognition by the mammalian innate immune system. The third Drosophila
Rel-homology domain protein, Relish, was also shown to be implicated in the
regulation of immune gene expression but is an activator of the other main
signaling pathway, the immunity-deficiency pathway (Imd pathway). Today,
many of the signal transducing components of the Toll- and Imd-pathways has
been characterized (Fig.1).
Figure 1. The Toll and Imd pathways.
The Toll pathway
The Toll signaling pathway and many of its components were characterized by
Kathryn Andersson and colleagues in their studies of embryonic polarity
establishment (reviewed in (Belvin and Anderson, 1996)). For immune gene
15
regulation, the pathway can be activated by receptor binding to bacterial
peptidoglycan from many Gram-positive species, or by interactions with some
species of fungi. For peptidoglycan activation the pathway requires
peptidoglycan recognition protein (PGRP)-SA and Gram-negative binding
protein (GNBP)-1, or PGRP-SD (Bischoff et al., 2004; Gobert et al., 2003;
Michel et al., 2001; Pili-Floury et al., 2004). PGRP-SA is presumably acting
together with Gram-negative binding protein-1 (GNBP1) in a recognition
complex but PGRP-SD is seemingly independent of GNBP1.
How the signal is transduced immediately after the recognition event
is not clear. It is, however, likely that serine proteases are activated, which
subsequently is leading to cleavage of the peptide-cytokine Spätzle to its active
form. Spätzle then bind to the Toll-receptor in the cytoplasmatic membrane of
fatbody cells. Binding of Spätzle to Toll induces receptor-dimerization and this
in turn leads to the recruitment of the adaptor protein MyD88 to the
cytoplamatic Toll-Interleukin-1 receptor (TIR) domains of Toll receptors (Sun et
al., 2004). MyD88 interacts with the Toll receptors via its own TIR domain, and
to the adaptor protein Tube and to Pelle via death domain interactions, and is
thus initiating the formation of a signaling complex. Pelle is a serine-threonine
kinase and upon activation it promotes phosphorylation of Cactus (an IκB
homolog), which is bound to the transcription factor Dif (an NFκB homolog).
Once phosphorylated, Cactus is targeted for degradation, and Dif is free to enter
the nucleus and start transcription (Wu and Anderson, 1998).
When the pathway is activated by fungi, the events that precedes
Spätzle cleavage is following a different route than while activated by bacteria.
The serine protease Persephone and the serine protease inhibitor Necrotic act
upstream of Spätzle (Levashina et al., 1999; Ligoxygakis et al., 2002a) but the
actual receptor(s) needed for pathway activation still remains to be found.
Even if the Toll pathway is thoroughly studied there are still some
question marks remaining. For example, it is not shown whether Pelle is actually
phosphorylating Cactus or if there is another kinase(s) between Pelle and
Cactus, and it is not clear if Dorsal is involved in immune gene activation, or if
Dif is the only Toll-mediated transcription activator.
The Imd pathway
The Imd-pathway has many similarities with the mammalian Tumor-necrosis
factor (TNF) receptor pathway. A major difference, however, is that the TNFreceptor itself is not present in the Imd pathway. Instead, membrane-spanning
PGRP-LC receptors activate the pathway (Choe et al., 2002; Gottar et al., 2002;
Ramet et al., 2002b) after binding to certain types of peptidoglycan (recognition/
activation specificities will be discussed later). These receptors have a PGRP
domain outside the cytoplasmatic membrane, a single membrane-spanning helix,
and a cytosolic ~30 kDa non-PGRP domain, which shares no significant
16
homology to any known protein. Downstream of PGRP-LC is the Imd protein
and the corresponding gene gave name to the pathway when a null-mutant was
found severely hampered in its antimicrobial peptide expression (Lemaitre et al.,
1995). Recently, the Imd protein was shown to interact with the non-PGRP
domain of PGRP-LC (Choe et al., 2005). The Imd protein has a C-terminal
Death domain that interacts with the cytosolic protein FADD and the caspase
Dredd. The N-terminal half of Imd interacts with the cytosolic non-PGRP
domain of the PGRP–LC receptors and is required for pathway activation. The
pathway has a branch-point downstream of the Imd protein and one branch
activates FADD – Dredd while the other branch activates TAK1. TAK1 is a
kinase and it is in turn constituting a new branch-point of which one branch
leads to the activation of the IKK complex and the other to the activation of the
Jun-N-terminal kinase (JNK) pathway controlling stress, and cellular immune
responses (Silverman et al., 2003). The IKK complex is composed of the two
subunits IKKβ (ird5) and IKKγ (kenny) (Lu et al., 2001; Rutschmann et al.,
2000) and when activated it promotes phosphorylation of the NF-κB homolog
Relish. Relish is a Rel-homology protein that has an N-terminal transcription
activating domain and an internal IκB-like domain at the C-terminus. When
phosphorylated, Relish is endoproteolytically cleaved (Stoven et al., 2000),
presumably by the caspase Dredd after which the N-terminal domain
translocates to the nucleus and initiates immune gene transcription.
Toll and Imd are the major immune pathways
How many signal transduction pathways are controlling the immune gene
expression in Drosophila? Flies that are simultaneously mutated in the Toll-,
and the Imd-pathway, are totally unresponsive in terms of anti-microbial peptide
gene expression. Such flies were the subject of a gene microarray study, in
which the immune response was compared to that of a wild-type fly and to flies
defect in only one of the pathways (De Gregorio et al., 2002b). It was shown
that the expression levels of nearly 300 genes were significantly affected
(induced or repressed) by a septic injury in the wild-type fly and that 70% of
these were affected in the mutant flies. Some genes are controlled by both
pathways but most of the genes are dependent on only one of the pathways.
Genes controlled by the Imd pathway includes most of the anti-microbial
peptide genes but also many other immune related genes such as enzymes
involved in the melanization process (Pale, Punch and Dhpr), prophenoloxidaseactivating enzyme (proPO-AE), and several PGRPs (-SD, -SB1, LB). Besides
drosomycin, the Toll pathway controls expression of several serine proteases,
serine protease inhibitors (serpins), and it also regulates the components of the
Toll-pathway itself. However, it was also found that some immune related genes
were not at all affected in the Toll/ Imd double mutants, suggesting that other
pathways also regulate expression of at least some immune gene. As described
17
in the following section, at least two other immunity-related pathways are
present.
Other pathways
In addition to the described NFκB signaling pathways there are at least two
other signaling pathways involved in Drosophila immunity, the Janus kinase
(JAK)/ signal transducers and activators of transcription (STAT) and the c-Jun
N-terminal kinase (JNK) pathways. The immunological outcome of these two
pathways is not as well-studied as that of the Toll and Imd pathways. In
response to septic injury the JAK/ STAT pathway controls the production of at
least two humoral factors synthesized in the fatbody, the thioester-contaning
protein 1 (Tep1) and Turandot A (TotA) (Agaisse et al., 2003; Lagueux et al.,
2000). Tep1 is a member of the complement C3/ α2-macroglobulin superfamily
which has not been thoroughly investigated in Drosophila, but in Anopheles
gambiae, the malaria vector, it has been shown to promote phagocytosis of
Gram-negative bacteria (Levashina et al., 2001). The role of the Torandot genes
is not known but their expression is upregulated in response to stress conditions
(Ekengren and Hultmark, 2001). The JAK/ STAT pathway is also involved in
blood cell proliferation and differentiation. The differentiation of hemocytes into
lamellocytes during a parasitic wasp infection is clearly linked to this pathway
(see (Agaisse and Perrimon, 2004) for a review). The JNK-pathway branches off
from the Imd pathway and controls cytoskeletal, and proapoptotic genes, and is
involved in wound healing (Boutros et al., 2002; Goberdhan and Wilson, 1998;
Ramet et al., 2002a).
In analogy with the Toll pathway both the JAK/ STAT and the JNK
pathways are, in addition to the immunological involvement, also involved in
the control of other processes during embryonic development, reviewed in (Luo
and Dearolf, 2001; Noselli, 1998).
Pattern recognition
For many decennia immunologists were focused on antigen recognition by the
adaptive immune system. In 1989, however, the so-called “pattern recognition
concept” was formulated by Charles Janeway Jr (Janeway, 1989). He
foresighted the existence of germ-line encoded pattern recognition receptors
(PRR) that had capacity to detect molecular patterns present uniquely in
microbial cells and consequently absent in the host. These “pathogen-associated
molecular patterns”, or PAMPs, should be essential building blocks of the
microbial cells and evolutionary conserved among large groups of microbes.
This strategy enables a small number of germ-line encoded receptors to
recognize an almost unlimited number of bacterial species. Eight year after
publishing his theory, Janeway together with Ruslan Medzhitov, were involved
in the discovery of the first pattern recognition receptor in mammalian host
18
defense, Toll-like receptor 4 (TLR4) (Medzhitov et al., 1997). This receptor was
soon shown to have capacity to recognize lipopolysaccharide (Poltorak et al.,
1998), a molecule present in the outer-membrane of Gram-negative bacterial
cells. LPS fulfills the criteria for a good PAMP as it is an essential structural
component of the outer membrane in Gram-negative bacterial cells. Moreover,
LPS has for long been known to elicit conditions like inflammation and fever in
mammals but the underlying mechanisms were still unknown. These initial
findings opened many closed doors and a hunt for related pattern recognition
receptors had started. Today, twelve different PRRs of two classes (TLRs and
NODs) are described in mammalian host defense and they have the amazing
capacity to recognize almost all different bacterial groups existing (reviewed in
(Philpott and Girardin, 2004). After bacterial detection, a proinflammatory
response is induced through NFκB activation, and the adaptive branch of the
immune system is initiated through crosstalk with dendritic cells (Pasare and
Medzhitov, 2004). The impact of the pattern recognition concept and discovery
of the innate immune receptors is now appreciated as being one of the most
important recent findings in the field of immunology. The term “pathogenassociated molecular patterns” should, however, not be associated with
pathogens in particular but rather with microbes in general.
Microbial immune targets
A molecule present on a microbe must fullfill some basic criteria for being a
good PAMP. It must be widespread among large groups of microorganisms and
it must be fairly invariant during evolution. Typically, PAMPs are essential
molecular building blocks of the microbial cell. In the following sections are
examples of such immune target molecules.
Teichoic acids
Teichoic acid (Fig. 2) is an anionic polymer of glycerol-phosphate or ribitiolphosphate that is interwoven in the cell wall of Gram-positive bacteria and
extends out and beyond the peptidoglycan layers. It can either be covalently
connected to the peptidoglycan or have a lipid part that is anchored in the
plasma membrane. Teichoic acids are functional analogs of lipopolysaccharide
and confer a net negative charge to the bacterial cell. The degree of negative
charge is modulated by the addition of protonated D-alanyl groups. Teichoic
acids affect the traffic of ions, nutrients, proteins, and antibiotics across the cell
wall (for an extensive review see (Neuhaus and Baddiley, 2003)).
19
Figure 2. Teichoic acid. Part of the polymeric molecule.
Lipopolysaccharide
The outer membrane of Gram-negative bacteria is a lipid bilayer, of which the
inner leaflet is a phospholipid monolayer and the outer leaflet is a monolayer of
lipopolysaccharide (LPS). The LPS molecule (Fig. 3) has a lipid part, lipid A.
Connected to Lipid A is a core region consisting of various saccharides
including 2-keto-3-deoxyoctulosonic acid (KDO), which is uniquely present
among bacteria. Extending outside the core region is a polysaccharide O-chain,
or O-antigen, which can be of variable composition and length. The composition
of the Lipid A part and the core region is fairly invariant between bacterial
species. It has for long been known that LPS induces inflammation and fever in
humans. The microbial signature properties of LPS is strong since it is a
ubiquitous and conserved molecule having unique features only found in
bacteria.
Figure 3. Structure of lipopolysaccharide, LPS.
Fungal β–1,3-glucan
Fungi form a diverse group of eukaryotic microorganisms, of which many
species can cause infections in both invertebrates like Drosophila and in
20
vertebrates. A common fungal cell-wall molecule is β-1,3-glucan, which is a
polymer of D-glucose in β(1,3) linkage with β(1,6) linked side chains of varying
length and distribution. The structure is similar to that of chitin, which consists
of D-glucose units coupled in β(1,4) linkage. Even if β-glucan is found in the
cell walls of different plants (e.g. algae), it is not present in animals, which
makes it a good target molecule for non-self recognition of fungal
microorganisms (Brown and Gordon, 2003).
Peptidoglycan
Peptidoglycan is a polymeric macro-molecule present in the cell wall of almost
all bacteria. It maintains the shape and gives tensile strength to the cell and it
confers tolerance to changes in the osmotic pressure. Peptidoglycan is a network
of unbranched glycan chains crosslinked with short peptides.
Figure 4. General structure of peptidoglycan. Two tetrapeptides are linking
glycan chains. A tetrapeptide is connected to the lactyl group of a MurNAc
residue and to an adjacent tetrapeptide, either directly, or via an interpeptide
bridge (shown as a dotted line).
The glycan chains are composed of alternating residues of GlcNAc and
MurNAc linked by β, 1→4 bonds (Fig. 4) and are typically 10 to 100
disaccharide units long (Harz et al., 1990). The peptide unit is composed of two
21
tetrapeptides (L-Ala – γ-D-Glu – DA – D-Ala) linked to the glycan chains via the
amino group of L-Ala to the lactyl group of the MurNAc unit. The tetrapeptides,
in turn, are linked to each other through the branching DA (diamino) group
containing residue to the D-Ala residue. The DA residue is most commonly an
L-Lys or a meso-diaminopimelic acid (DAP). Sometimes the two tetrapeptides
are not directly linked but instead connected via an interpeptide bridge
(Schleifer and Kandler, 1972).
The general structure of peptidoglycan is much conserved among
bacteria and the variation that is lies mainly in the tetrapeptide linkage region.
The main differences are the identity of the third branching amino acid and the
existence of an interpeptide bridge. However, the variation is rather small and
large groups of bacteria have the same composition. Most common is the direct
linked DAP-type peptidoglycan which is present in probably all Gram-negative
species and in species belonging to Bacillaceae, Lactobacillaceae,
Corynebacteriaceae, and Propionibacteriaceae. The second most common
peptidoglycan type is those with an L-lysine residue in the third position of the
tetrapeptide. Lys-type peptidoglycans are more heterogeneous because they
often have an interpeptide bridge of variable but species-specific composition.
For example, in Staphylococcus aureus the interpeptide bridge is composed of
five glycine residues (Schleifer and Kandler, 1972).
Besides these two major groups, DAP- and Lys-type peptidoglycan,
other peptidoglycan variations include L-Orn, L,L-DAP, and hydroxyl-Lysine in
the third tetrapeptide position. In rare cases the tetrapeptide linkage is instead at
the D-Glu in second position. Peptidoglycan can be further modified. A common
modification is amidation of carboxyl groups of meso-DAP or D-Glu and a less
common is O-acetylation of muramic acid residues. The latter modification
confers resistance to some peptidoglycan hydrolyzing enzymes (Bera et al.,
2005; Schleifer and Kandler, 1972).
The composition of the Gram-positive and Gram-negative cell wall is
rather different. Gram-positive bacteria have a thick cell wall made up of 20 to
80 layers of peptidoglycan. In addition to peptidoglycan, the cell wall contains
proteins and teichoic acids. In Gram-negative bacteria, the peptidoglycan is only
one to three layers thick and is located in the periplasmatic space between the
plasma membrane, and the outer membrane. The peptidoglycan layer is
connected to the outer membrane by the covalently linked Brauns lipoprotein,
which is imbedded in the inner leaflet of the outer membrane.
The bacterial signature properties of peptidoglycan are indeed strong.
MurNAc is a hexose, which is only present in bacteria. The presence of D-amino
acid residues is also very rare among eukaryotes and the alternating motif of Dand L-amino acid residues in the peptidoglycan tetrapeptides is a feature unique
to bacteria. The MurNAc – tetrapeptide motif can thus be considered as a
bacterial bar-code and as such a perfect target for pattern recognition receptors.
22
Peptidoglycan synthesis and turnover
When rod-shaped bacteria grow, the peptidoglycan sacculus is constantly rebuilt
to enable elongation. In Gram-positive bacteria the cell wall biogenesis and
turnover result in a shredding of peptidoglycan fragments to the environment. In
most Gram-negative bacteria, on the other hand, an efficient peptidoglycan
recycling pathway ensures that only 5% of the peptidoglycan fragments are
released from the cell (Goodell and Schwarz, 1985; Greenway and Perkins,
1985; Park, 1995). There is an extensive peptidoglycan turnover during
exponential growth whereby nearly half of the peptidoglycan is degraded and
recycled to allow for the building of new glycan strands cross-linked with
peptides. Peptidoglycan biosynthesis is an intricate process and the generally
accepted model for Gram-negative bacteria invokes multimeric enzyme
complexes comprising peptidoglycan synthesizing and degrading enzymes
(Holtje, 1996b).
Figure 5. Simplified view of the “3 for 1” model proposed for Gram-negative
bacterial peptidoglycan biosynthesis. A multimeric enzyme complex digests one
glycan strand and synthesizes three new strands in place. Dark-grey circles
represent endopeptidases and lytic transglycosylases, which digest peptidoglycan and release the recycling fragments (the double-hexagone containing
objects). The white ellipse and light grey circles represent glycosyltransferases
and transpeptidases that incorporate new material transported from the
cytoplasm. The figure is adapted from (Holtje, 1996a).
23
The synthesizing enzymes include transferases and high-molecular weight
penicillin-binding proteins whereas the degrading enzymes comprise
endopeptidases and lytic transglycosylases. The model for peptidoglycan
synthesis (Fig. 5) suggests that one glycan strand is being removed while three
new strands are being synthesized in place (Holtje, 1996a; Koch, 1995). The
penicillin-binding proteins/ transpeptidases and glycosyltransferases incorporate
peptidoglycan precursor molecules at the sites which have been made available
by the action of the degrading enzymes. The glycan chains typically have a
MurNAc residue in 1,6 anhydro conformation at the reducing end (Vollmer and
Holtje, 2004). This feature is characteristic for Gram-negative bacterial
peptidoglycan and is a consequence of lytic transglycosylases cleaving the
glycan stand between MurNAc and GlcNAc residues. The major product
produced by the digestion/ synthesizing machinery is a GlcNAc-(1,6anhydro)MurNAc-tetrapeptide unit (Fig. 6)(Park, 2001).
Figure 6. Structure of the major peptidoglycan recycling molecule of Gramnegative bacteria, the GlcNAc-(1,6-anhydro)MurNAc-tetrapeptide, also known
as tracheal cytotoxin, TCT.
24
This molecule is recycled in a controlled process (Fig. 7) and its initial uptake
from the periplasm and transport into the cytoplasm is facilitated by a membrane
permease, in E. coli named AmpG (Cheng and Park, 2002). In the cytoplasm the
sugar moieties are cleaved off from the stem peptide by AmpD, a bacterial Nacetylmuramoyl L-alanine amidase (Jacobs et al., 1995). The tetrapeptide is then
further processed to a tripeptide, and then rebuilt to a UDP-MurNAcpentapeptide (Uehara and Park, 2004).
Figure 7. Peptidoglycan recycling and synthesis in Gram-negative bacteria.
During synthesis part of the pre-existing peptidoglycan is digested into TCT
(GlcNAc-(anhydro)MurNAc-tetrapeptides), which constitute the major turnover
product. This fragment is transported to the cytoplasm via the permease AmpG.
In the cyotosol AmpD cleaves off the two sugar moieties and further processing
yields an L-Ala – γ-D-Glu – mesoDAP tripeptide. The tripeptide is successively
transformed into UDP-MurNAc-pentapeptide with aid of MurF. This precursor
molecule is transported across the membrane and utilized for de novo
peptidoglycan biosynthesis.
Even though the turn-over products are efficiently taken back to the cytoplasm a
small portion (~5%) of the recycling-product is typically lost to the environment
during growth (Park, 1995). Interestingly, peptidoglycan fragments has for long
been reported to elicit a range of immunity related effects in humans including
adjuvanticity, cytotoxity, induction of arthritis, and modulation of sleep
(reviewed in (Boneca, 2005)). A suggestion presented in this thesis is that such
25
peptidoglycan fragments are major regulators of insect immunity as well. Some
bacteria release peptidoglycan fragments more actively, e.g. Neisseria
gonorrhoeae and Bordatella pertussis secret vast amounts of the GlcNAc – (1,6anhydro)MurNAc – tetrapeptide (Cundell et al., 1994; Melly et al., 1984). For
these bacteria the peptidoglycan fragment is a virulence factor that inflicts
damage to cells of the fallopian tubes (Neisseria) or to ciliated epithelial cells in
the respiratory tract (Bordetella). In the latter case the peptidoglycan fragment is
more known as tracheal cytotoxin (TCT) (Fig. 6) and is the major causative
agent for the symptoms of whooping cough.
It is possible that there exist a selective interplay between the host
innate defense and the peptidoglycan biosynthesis machinery of pathogenic
bacteria. A fact that speaks in favor for this is that some pathogens are
modifying their peptidoglycan making it resistant to host lysozyme. A
pathogenic strain of Streptococcus pneumoniae, for example, has been shown to
modify the GlcNAc units in the glycan back-bone by deacetylation. This
modification confers resistance to lysozyme and was shown to depend on one
single gene (pgdA) (Vollmer and Tomasz, 2002). In another example, AmdD
mutants of Salmonella enterica serovar Typhimurium that are accumulating
TCT in the cytosol, are less prone to invade macrophages and are better inducers
of iNOS than wildtype bacteria. This suggests that the peptidoglycan recycling
pathway is involved in virulence (Folkesson et al., 2005).
26
Figure 8. Peptidoglycan digesting enzymes. The general structure of Gramnegative bacterial peptidoglycan is shown and arrows indicate which bonds are
cleaved by the different enzymes.
Peptidoglycan hydrolyzing enzymes
Below follows a brief overview of peptidoglycan degrading enzymes present in
bacteria and some examples of viral and eukaryotic counterparts.
β-N-acetylmuramidase: Hydrolyses the β(1,4) bond between MurNAc and
GlcNAc in the glycan chains and is involved in bacterial autolysis (Shibata et
al., 2005; Turner et al., 2004). This enzyme type to which hen egg white
lysozyme belongs is widely spread among eukaryotes. In humans, it is present in
high concentrations in sera and saliva and participates in immune defense
reactions (Vanderwinkel et al., 1995).
β-N-acetylglucosaminidase: Hydrolyses the β(1,4) bond between GlcNAc and
MurNAc in the glycan chains. In E. coli NagZ is a cytosolic enzyme involved in
peptidoglycan recycling (Cheng et al., 2000; Votsch and Templin, 2000).
Lytic transglycosidase: Cleaves the same β-glycosidic bond as β-Nacetylmuramidase but in addition concomitantly forms a 1,6-anhydromuramoyl
residue. Works in concert with a D,D-endopeptidase in the digestion/
synthesizing enzyme complex to produce the GlcNAc – (1,6-anhydro)MurNActetrapeptide recycling product. The enzyme is present in most bacteria except for
Gram-positives that only rarely have the enzyme for use in the sporulation
27
process (Atrih and Foster, 2001). This enzyme type has to my knowledge not
been reported from any eukaryote organism.
N-acetylmuramoyl L-alanine amidase (NAMLAA): AmpD in E. coli is
involved in the recycling-process and is present in the cytoplasm (Jacobs et al.,
1995) where it cleaves the amide bond that unites the stempeptide and the
glycan portion in the recycled molecule. Bacteriophage T7 lysozyme is another
example that utilizes the enzyme to lyse host cells and facilitate phage escape
from infected E. coli cells (Cheng et al., 1994). NAMLAA enzymes will also be
the subject of papers I and II.
D,D-carboxypeptidase: Engaged in the peptidoglycan synthesis process.
Cleaves off the terminal D-Ala from the GlcNAc – MurNAc - pentapeptide
precursor molecule to facilitate incorporation and transpeptidation to an adjacent
tetrapeptide.
L,D-carboxypeptidase: Cleaves the bond between the meso-DAP and the
terminal D-Ala in the same tetrapeptide. Cytoplasmic LdcA is involved in
peptidoglycan recycling (Templin et al., 1999). PGRP-SA in Drosophila is an
example of a eukaryotic protein with this enzyme activity and it has possibly an
immunomodulatory function (Chang et al., 2004).
D,D-endopeptidase: Cleaves the bond between the mesoDAP of one
tetrapeptide and the terminal (fourth) D-Ala of the adjacent tetrapeptide. Works
in concert with lytic transglycosylases in the digestion/ synthesizing enzyme
complex to produce the GlcNAc-(1,6-anhydro)MurNAc-tetrapeptide recycling
product (Holtje, 1996b; Romeis and Holtje, 1994a; Romeis and Holtje, 1994b).
Lysostaphin is an example of another type of endopeptidase that is active on
Staphylococcal peptidoglycan and has specificity for the Gly – Gly bonds of the
pentaglycine interpeptide bridge.
Recognition molecules
This section gives a brief description of some of the most important groups of
innate immune receptors from both vertebrates and invertebrates including a
more thoroughly description of the peptidoglycan recognition proteins.
C-type Lectins
Lectins can bind to sugar residues of different kinds. Specifically engaged in
immune defense are the C-type lectins, which have affinity for sugars that are
common among microbes but rare in the host. Mannan-binding lectin is a
vertebrate serum protein with collagen like thread structures and with
carbohydrate recognition domains at the ends. It binds to bacterial surface sugar
residues such as mannose, fucose, N-acetylglucosamine (Ma et al., 2004).
Ligand binding activates associated serine proteases which in turn initiates the
recruitment of complement factors. Dectin-1 is another example of a C-type
28
lectin that recognizes fungi by binding to β-glucan. After binding it associates
with TLR-2 to induce a NF-κB response (Brown et al., 2003; Gantner et al.,
2003). An example of an invertebrate C-type lectin is the immunolectin-2 of the
tobacco hornworm Manduca sexta, which specifically binds to LPS and initiates
the prophenoloxidase cascade (Yu and Kanost, 2004).
Toll-like receptors
The Toll-like receptors (TLRs) has already been introduced in a previous
section. For a more extensive review of TLRs including ligand specificity I refer
to (Takeda and Akira, 2005). There are ten TLR homologues in the human
genome data base and they all encode integral membrane proteins. The
extracellular domain consists of leucine-rich repeats (LRR) and the cytoplasmic
domain, called the Toll/ IL-1 receptor (TIR) domain, associates with adapter
proteins of the NF-κB signaling pathway. The TLR can either be anchored in the
plasma membrane or in the membrane of intracellular endosomes. The LRR
domains interact with the bacterial associated ligands and each TLR displays its
own unique affinity. Additional co-factors are in some cases also required for
recognition. The LPS receptor TLR-4 for example, is dependent on the adaptor
proteins CD14 and MD-2 to be fully active (da Silva Correia et al., 2001). In
some cases the recognition event requires receptor heterodimerization. TLR-2
associates with both TLR-1 and TLR-6 to detect lipopeptides (Takeda et al.,
2002). TLR-3, -7, -8, and -9 are associated with endosomes and recognizes
bacterial or viral nucleic acids (Alexopoulou et al., 2001; Heil et al., 2004;
Hemmi et al., 2000). TLR-2 was initially thought to be a peptidoglycan receptor
but it is now believed to recognize lipoteichoic acids and lipoproteins
(Hirschfeld et al., 1999; Hirschfeld et al., 2000; Schwandner et al., 1999).
Not only are the TLRs mediating an innate immune response but they
are also cross-talking with the adaptive branch of the immune system. Especially
dendritic cells are engaged in this cross-talk since they process/ present antigen
and express TLR-mediated co-stimulatory molecules (Takeda and Akira, 2005).
Nod receptors
The nucleotide-binding oligomerisation domain (Nod) proteins are cytosolic
peptidoglycan receptors of the vertebrate innate immune system. They consists
of a C-terminal LRR domain, a central nucleotide binding domain, and one
(Nod1) or two (Nod2) caspase-activating and recruitment domains at the Nterminus. Both Nod1 and Nod2 recognize peptidoglycan but they differ in the
specific epitope requirement. Nod1 binds the γ-D-Glu-meso-DAP dipeptide
whereas Nod2 instead recognizes a MurNAc-L-Ala-γ-D-Glu subunit. These
specificities define Nod2 to be a more general receptor and Nod1 to be restricted
to sense bacteria having DAP-type peptidoglycan. Upon ligand binding Nod
29
receptors, just like TLRs, triggers an NFκB response. Interestingly, mutations in
Nod2 have been implicated Crohn’s disease, a chronic inflammatory disease in
the intestine, and in the Blau syndrome, which is an inflammatory disease
affecting joints and eyes (see (Philpott and Girardin, 2004; Royet and Reichhart,
2003) for reviews on Nods).
Gram-negative binding proteins
The Gram-negative binding protein (GNBP)/ β-glucan recognition protein
family was originally discovered in lepidopteran species where protein members
were isolated and characterized biochemically (Ma and Kanost, 2000; Ochiai
and Ashida, 1988). The proteins contain an N-terminal glucan binding domain
and a C-terminal domain that shares homology to bacterial glucanases.
However, the catalytic amino acid residues required for enzymatic activity are
not retained (Zhang et al., 2003). Members of this protein family have been
reported to bind LPS in Bombyx mori and β-1,3-glucan M. sexta. The
Drosophila genome comprises three GNBP members and one of these, GNBP1,
is part of the Toll pathway (Gobert et al., 2003; Kim et al., 2000; Pili-Floury et
al., 2004). Somewhat surprising is that even though this protein has affinity for
both LPS and β-1,3-glucan it is required for the induction with Gram-positive
bacteria, which do not contain these molecules. However, it is not clear if the
protein takes part in the recognition event or if it is mediating the signal from the
PGRP-SA receptor.
The expression of a GNBP protein has been found specifically
uppregulated in the gut region of A. gamiae in response to a Plasmodium
falciparum infection (Tahar et al., 2002).
Peptidoglycan recognition proteins
PGRP is the most studied pattern recognition receptor in insects. The following
sections surveys the PGRP research of past and present.
The PGRP-story of our laboratory starts in 1995 when Håkan Steiner
and Daiwu Kang, a Chinese PhD student, decided to investigate how insects
defend themselves against virus infections. They planned to set up a differential
display screen with the aim to identify the genes that were up- and downregulated by viral infections. For training purposes a screen was initiated using
bacteria instead of viruses and larvae from the Lepidopteran species
Trichoplusia ni were infected. Differentially expressed were identified using
data-base mining and homology searches (Kang et al., 1996). One of the cloned
genes had similarities a protein from the silk moth B. mori. A few N-terminal
residues of had been sequenced of this protein and it was named peptidoglycan
recognition protein (PGRP) (Yoshida et al., 1986). This protein could bind
30
peptidoglycan and was required for prophenoloxidase cascade activation. A
similar protein could be a good candidate also for a receptor controlling
antimicrobial peptide genes. The interest in PGRP increased when the laboratory
cloned homologous genes from mouse and man. The T. ni PGRP and a mouse
homolog was expressed and purified and both proteins did show peptidoglycan
affinity (Kang et al., 1998). Since mice are devoid of the PPO cascade, the
function of at least that particular PGRP variant could not be the same as in B.
mori. It was found that PGRP is anciently related to bacteriophage T7 lysozyme,
a peptidoglycan degrading enzyme. However, no such activity could be recorded
for the mouse or the T. ni PGRP. The lack of enzymatic activity pointed instead
towards a possible receptor function for these proteins. Daiwu Kang also cloned
a PGRP homolog from Drosophila melanogaster, later named PGRP-SA. The
flanking sequence placed the gene adjacent to an RNA polymerase in the
genome. Collaboration was initiated with Dan Hultmark’s group in Umeå to
make a PGRP mutant fly. However, it was soon evident that the Drosophila
genome contains several PGRP genes and the risk was therefore obvious that a
knock-out of a single PGRP would lack a phenotype. Instead, a general
characterization of all the Drosophila PGRPs was performed including tissue
distribution and possible inducibility by bacterial fragments (Werner et al.,
2000).
PGRP in Drosophila: Expression pattern and protein topology predictions
When the Drosophila genome was completely sequenced in 2000, it was found
to harbor a total of 13 PGRP genes (Khush and Lemaitre, 2000). They have been
grouped into two groups according to the length of their transcripts, the short
(S), and the long (L) forms (Werner et al., 2000). There are seven short forms
which all encode proteins that contain the conserved PGRP domain flanked by
an N-terminal signal peptides that target these proteins for cellular export. The
short forms include PGRP-SA, -SB1, -SB2, -SC1A, -SC1B, -SC2, and -SD. The
majority of the short PGRPs are expressed in the fatbody and the expression is
often up-regulated upon bacterial infection. The N-terminal signal sequence and
the fatbody expression strongly suggest that these proteins are secreted into the
hemolymph. Some short forms (PGRP-SC) are also expressed in the gut region.
An export to the gut lumen is likely and the proteins are possibly taking part in
bacterial decomposition. Another possibility is that the proteins take part in the
defense of the barrier epithelium lining the gut.
The longer forms, which typically contain another domain in
addition to the PGRP part, include PGRP-LA, -LB, -LC, -LD, -LE, and -LF. The
additional non-PGRP domains differ in amino acid length and they neither share
homology to each other nor to any other protein. The amino acid sequences of
PGRP-LA, -LC, and -LF contain putative membrane spanning regions and
intracellular non-PGRP domains, in addition to the extracellular PGRP domain.
31
Membrane localizations for these forms are therefore likely and have been
experimentally shown for PGRP-LC (Paper IV, V). PGRP-LF is unique since it
is the only Drosophila PGRP that contains two PGRP domains, located in
tandem outside the putative transmembrane domain.
Topology predictions for the other long forms are more uncertain.
The cDNA for PGRP-LB encodes a short protein more similar to the short (S)
forms. However, it does not have any obvious signal peptide sequence and was
therefore initially predicted to be an intracellular protein (Werner et al., 2000).
The protein sequences of PGRP-LD, and –LE, both contains a non-PGRP part
connected to the amino-terminal of the PGRP domain, and also for these
proteins are no signal peptide present. However, an investigation shows that
when PGRP-LE is overexpressed, the recombinant protein is anyhow secreted to
the hemolymph (Takehana et al., 2002).
Functional studies of Drosophila PGRPs
The capacity of PGRP to bind the bacterial cell wall polymer peptidoglycan
points by itself toward functions involved in immune reactions. Also the
bacteria-inducible expression patterns of most PGRPs and the localized
expression in immune related tissues strongly corroborates an involvement in
the immune defense (Werner et al., 2000).
The first Drosophila PGRP to be assigned a function was PGRP-SA.
It was found that the protein was required for Toll pathway activation (Michel et
al., 2001). Epistatic gene knockout experiments placed PGRP-SA upstream of
Toll and showed that it is required for down-stream processing of the peptide
cytokine Spätzle, presumably by activating a series of serine proteases. PGRPSA is thus a soluble receptor involved in recognition of certain types of Grampositive bacteria such as Micrococcus luteus and Streptococcus faecalis and
knock-out flies are more susceptible to infections by these bacteria. PGRP-SA
has later been suggested to be engaged in an extracellular complex-formation
with GNBP-1 (Gobert et al., 2003; Pili-Floury et al., 2004). Moreover, PGRPSA have been found to possess L,D-carboxypeptidase enzymatic activity (Chang
et al., 2004). This might have an immunomodulatory function important for
Toll-pathway signaling but its particular role needs to be further investigated.
PGRP-SD is another short form that has also been reported to be an
extracellular factor of the Toll pathway (Bischoff et al., 2004), in parallel to
PGRP-SA and GNBP-1. The PGRP-SD mutant is hampered in the response to
infections of S. aureus and Streptococcus pyogenes and shows partial
redundancy with PGRP-SA/ GNBP1.
PGRP-SC1B, and –SB1, are characterized in Paper I and II,
respectively, and will be discussed later in this thesis. No functional study has
yet been done for the short forms PGRP-SB2, SC1A, and SC2, but they are
further discussed in Paper I and II.
32
PGRP-LC was the first of the long membrane-bound forms to be
functionally characterized. It was shown to be a receptor of the Imd-pathway
upstream of the Imd protein and was absolutely required for pathway activation.
This finding was reported in three independent studies in April 2002 (Choe et
al., 2002; Gottar et al., 2002; Ramet et al., 2002b). The PGRP-LC knockout flies
were highly susceptible to Gram-negative bacterial infections. One of the studies
(Ramet et al., 2002b), showed that RNAi knock-down of PGRP-LC expression
impaired a Drosophila Scheider-2 cell line in the phagocytic response against E.
coli cells. The PGRP-LC gene locus is somewhat complex and three different
gene products can be formed by alternative splicing. The explicit role of the
different spliceforms as Imd-receptors, and biochemical data on peptidoglycan
affinities is the subject of Paper III, IV, and V.
PGRP-LE has also been reported to function as a receptor for the
Imd-pathway and genetic epistasis experiments place the protein, somewhat
remarkable, both upstream and in parallel with PGRP-LC (Takehana et al.,
2004). PGRP-LE binds DAP-type peptidoglycan and its overexpression in
Drosophila larvae leads to melanin formation which implies a possible role as a
receptor also for the PPO cascade (Takehana et al., 2002).
PGRP-LB was the first PGRP for which the crystal structure was
solved. It was also shown to possess N-acetylmuramoyl L-alanine amidase
activity and is thereby a peptidoglycan degrading enzyme (Kim et al., 2003). No
other functional study of PGRP-LB has been carried out.
The possible involvement of PGRP-LA and PGRP-LD in Imdpathway activation or phagocytosis of bacteria has been investigated using
RNA-interference (RNAi) to knock-down their expression. However, no clear
phenotypes could be detected (Ramet et al., 2002b).
RNAi against PGRP-LF gives a stronger Imd-mediated expression
of antibacterial peptide genes upon bacterial challenge (Paper III and Carina
Persson, personal communication). This suggests that the protein has an
inhibitory effect to the Imd-pathway.
PGRPs in other insects
Drosophila melanogaster is by far the most studied insect. The understanding of
its immune system is un-matched in any other insect species. Even though
PGRP genes and proteins have been characterized in other insect species, it is
not known if they play as central roles as they do in Drosophila. The malaria
vector Anopheles gambiae that belongs to the same insect order as Drosophila
(Diptera) harbors seven PGRP genes, of which many isoforms are remarkably
similar to their Drosophila counterparts. A striking example is the PGRP-LC
locus in which the organization of the exons producing three different
spliceforms is fully conserved (Christophides et al., 2002).
33
As already mentioned, the first PGRP that was characterized was
purified from hemolymph of the silk moth B. mori by Ashida’s group (Yoshida
et al., 1996; Yoshida et al., 1986). They found that this 19 kDa protein binds to
peptidoglycan and is required for peptidoglycan-induced activation of the prophenoloxidase cascade.
The first PGRP cDNA was cloned from the cabbage looper T. ni and
was performed in our laboratory (Kang et al., 1998). The gene showed high
sequence similarity to bacteriophage T7 lysozyme and the protein has
peptidoglycan affinity but no enzymatic activity. The functional role for the T. ni
PGRP is still not fully understood.
A PGRP was isolated from the hemolymph of the large beetle
Holotrichia diomphalia and was found to bind both peptidoglycan and the
fungal cell wall component β-1,3-glucan (Lee et al., 2004). Interestingly, it was
shown to be required for pro-phenoloxidase activation by β-1,3-glucan but not
by peptidoglycan.
Mammalian PGRPs
It was realized at an early stage of PGRP research that PGRP homologues were
also present in mammalian genomes (Kang et al., 1998). Now when the human
genome has been fully sequenced it is evident that it contains a total of four
PGRP homologues. There is one short (S) form, one long (L) form, and two of
intermediate (I) size (Dziarski, 2004).
The PGRP-S is most prominently expressed in the bone marrow and
in neutrophils. In murine neutrophils, PGRP-S is contained within intracellular
vesicles and has been shown to inhibit growth of Gram-positive bacteria (Liu et
al., 2000). A recent study (Cho et al., 2005) shows that also the human PGRP-S
is localized in neutrophil granules and accordingly inhibits growth of both
Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). In
addition, this protein shows synergistic antibacterial activity with lysozyme. A
Russian research group has found PGRP-S expressed in murine tumors and
suggested a cytokine function for the protein (Kiselev et al., 1998) and later they
reported that it can form a cytotoxic complex together with heat-shock protein
70 (Sashchenko et al., 2004).
The PGRP-Iα and PGRP-Iβ are putative membrane bound proteins
with a rather short intracellular part. Both these proteins have a localized
expression in the esophagus epithelium (Liu et al., 2001) of which relatively
little is known in terms of innate immune recognition and defense. No function
has yet been suggested for these proteins. However, the crystal structure of
PGRP-Iα has been solved and will be discussed in a section below.
PGRP-L contains a relatively large non-PGRP domain (~40 kDa)
with two hydrophobic stretches that weakly indicates putative membrane
spanning domains. It was therefore suggested to be a membrane bound protein
34
(Dziarski, 2004) and as such considered a strong immune receptor candidate.
However, it is pre-dominantly expressed in the liver and has later been found to
be an enzyme with peptidoglycan degrading activity (Gelius et al., 2003; Wang
et al., 2003). It was also realized that PGRP-L is identical to the Nacetylmuramoyl L-alanine amidase, or NAMLAA, that had been purified from
human sera and biochemically characterized in 1980s and -90s (Hoijer et al.,
1998; Hoijer et al., 1997; Hoijer et al., 1996; Mollner and Braun, 1984; Valinger
et al., 1982). These findings was recently reconfirmed (Zhang et al., 2005).
A recent study showed that in addition to the tissues showing major
PGRP expression, the expression of all four PGRPs could be markedly induced
in an NF-κB dependent manner in oral epithelial cells when challenged with
synthetic TLR- or Nod-ligands (Uehara et al., 2005). This tissue is a major site
for bacterial entrance to our bodies and it typically harbor bacterial
communities. However, not much is known of the interplay between these
bacteria and the innate immune system.
A bovine PGRP ortholog was isolated from neutrophil and
eosinophil granules and was reported to possess antibacterial activity against
Gram-positive and Gram-negative bacteria and surprisingly also against yeast
(Tydell et al., 2002). Since yeast does not contain peptidoglycan, this protein
was instead named bovine oligosaccharide-binding protein, bOBP.
A camel PGRP has been isolated from milk and was suggested to
function as an antibacterial factor (Kappeler et al., 2004). A rat PGRP has been
found up-regulated when animals have been deprived from sleep (Rehman et al.,
2001). Sleep-depravation can also be induced by peptidoglycan fragments
(Johannsen et al., 1990). A functional role for the rat PGRP as a modulator of
peptidoglycan-induced sleep deprivation was suggested (Rehman et al., 2001).
PGRP structures
The crystal structures of the Drosophila PGRP-LB and PGRP-SA and the
human PGRP-IαC and PGRP-S have been solved (Chang et al., 2004; Guan et
al., 2004a; Guan et al., 2004b; Guan et al., 2005a; Guan et al., 2005b; Kim et al.,
2003). They all have a similar fold as phage T7 lysozyme (Cheng et al., 1994),
which was expected considering the considerable amino acid sequence
similarity. The general structure contains three α-helices and a central β-sheet,
composed of four parallel strands and one anti-parallel strand (Fig. 9). However,
PGRP-SA has some additional minor helices and β-strands (Chang et al., 2004).
They all share a similar peptidoglycan-binding groove with the beta-sheet as a
floor and with walls flanked by one of the helices and five of the loop-regions.
The enzymatic PGRP-LB has, just like phage T7 lysozyme, a zinc ion
bound to one cysteine and two histidine residues in the catalytic centre. The
35
metal co-factor is not included in the other PGRP structures. Common to all
PGRP structures, but not present in T7 lysozyme, is an extra loop-region located
on the back of the protein relative to the binding cleft. It has been suggested that
this part could be important for protein – protein interactions and thus might
facilitate PGRP dimerization or complex-formation such as that suggested for
PGRP-SA and GNBP-1.
Adapted from Kim et al. Nature Immunology (2003) 4,787-793
Figure 9. Figure show ribbon diagrams of PGRP-LB (left) and bacteriophage
T7 lysozyme (right) structures. The zinc ion is symbolized by a ball, disulphide
bonds shown in “ball and stick”, and PGRP specific segment in yellow.
Also in contrast to T7 lysozyme, all PGRP structures contain disulphide bridges,
of which at least one is seemingly conserved in all PGRPs except in Drosophila
PGRP-LE (Guan et al., 2005b). This feature speaks against the proposed
cytoplasmatic localization of some PGRPs lacking an N-terminal signal-peptide,
because the reducing cytosolic environment will most likely disrupt disulphide
bonds.
36
Protein
D.m.PGRP-SA
D.m.PGRP-SB1
D.m.PGRP-SB2
D.m.PGRP-SC1A
D.m.PGRP-SC1B
D.m.PGRP-SC2
D.m.PGRP-SD
D.m.PGRP-LA
D.m.PGRP-LB
D.m.PGRP-LCa
D.m.PGRP-LCx
D.m.PGRP-LCy
D.m.PGRP-LD
D.m.PGRP-LE
D.m.PGRP-LF
B.m.PGRP
H.d.PGRP
C.d.PGRP
B.t.PGRP,
bOBP
R.n.PGRP
M.m.PGRP-S
M.m.PGRP-L
H.s.PGRP-S
H.s.PGRP-Iα
H.s.PGRP-Iβ
H.s.PGRP-L
Function
Receptor of Toll pathway, L,D-carboxypeptidase
activity.
N-acetylmuramoyl
L-alanine
amidase
(NAMLAA), antibacterial activity.
Putative NAMLAA, mainly expressed in prepupal stage.
Putative NAMLAA.
NAMLAA, peptidoglycan scavenger
Putative NAMLAA.
Extracellular factor of the Toll pathway, putative
receptor.
Function unknown, putative membrane protein.
NAMLAA.
Putative adapter-protein to PGRP-LCx required
for
Imd-activation
with
monomeric
peptidoglycan.
Main receptor of Imd-pathway.
Function unknown, membrane bound.
Function unknown.
Receptor of Imd-pathway and possibly involved
in PPO activation.
Function unclear, negatively affects Imd-pathway
signaling, membrane bound, protein contains two
PGRP-domains.
Receptor for PPO cascade activation
Ref
(Chang et al., 2004;
Michel et al., 2001)
II
I
I
I, (Gelius et al., 2003)
I
(Bischoff et al., 2004)
(Werner et al., 2000)
(Kim et al., 2003)
III,IV,V
III,IV,V
III,IV
(Werner et al., 2000)
(Takehana et al., 2002;
Takehana et al., 2004)
III,
Carina
Persson
(personal comm.)
(Yoshida et al., 1996;
Yoshida et al., 1986)
Receptor for PPO cascade activation, binds β- (Lee et al., 2004)
glucan and peptidoglycan
Present in milk
(Kappeler et al., 2004)
Antibacterial against Gram-positive and Gram- (Tydell et al., 2002)
negative bacteria and fungi
Suggest involvement in sleep deprivation (Rehman et al., 2001)
responses
Bacteriostatic. Expressed in neutrophil granula
(Liu et al., 2000)
NAMLAA
(Gelius et al., 2003)
Bacteriostatic. Expressed in neutrophil granula
(Cho et al., 2005)
Function unknown. Expressed in esophagus
(Liu et al., 2001)
Function unknown. Expressed in esophagus
(Liu et al., 2001)
NAMLAA. Mainly expressed in liver. Present in (Wang et al., 2003)
sera.
Table 1. Overview of the functional properties of PGRPs. D.m. Drosophila melanogaster
(Fruit fly), B.m. Bombyx mori (Silk moth), H.d. Holotrichia diomphalia (Korean Large
beetle), C.d. Camelus dromedaries (Camel), B.t. Bos Taurus (Cow), R.n. Rattus norvegicus
(Rat), M.m. Mus musculus (Mouse), H.s. Homo sapiens (Human).
37
The present study
Aim
The general aim of this thesis has been to investigate how the innate immune
system recognizes bacteria to initiate immune gene expression using Drosophila
as a model system. In particular, the involvement of peptidoglycan recognition
proteins, PGRPs, as immune receptors was the subject of the investigation.
However, the goal was extended during the course of the study to include the
characterization of enzymatic PGRPs.
A PGRP enzyme (Paper I)
The first project was to express and purify recombinant Drosophila PGRP
proteins and investigate their possible involvement as immune receptors or
modulators of immune gene expression. At this time, many of the intracellular
components of the Toll and Imd pathways were well-characterized but a
receptor triggering the pathways had yet not been found. The Toll receptor itself
was clearly not a bacterial receptor in striking contrast to the mammalian Tolllike receptors (TLRs), which had been shown to bind a repertoire of different
microbial ligands. We imagined that the soluble Drosophila PGRPs could either
function as opsonins that bind to bacteria and stimulate phagocytosis, or else
that they could function as co-receptors to a membrane receptor, e.g. Toll. A
similar function had been reported for the mammalian CD14 and LBP that binds
LPS and work as co-receptors for TLR 4 (Guha and Mackman, 2001).
One of our strategies was to assay immune gene induction in the
Drosophila S2 cell line after challenge with bacterial products and to assay the
expression of antibacterial peptide genes after addition of PGRP to the cell
culture medium. Initial studies by Gang Liu had shown that pretreatment with
PGRP-SA had an inhibitory effect on cecropin expression instead of the
elevated expression that was expected if the protein had been a receptor (the
result is fully explainable in light of later findings). To compare the inhibitory
effect of PGRP-SA with another soluble PGRP, a cell line expressing PGRPSC1B was established. When I finally had expressed and purified enough
protein to perform comparative studies with PGRP-SA, a paper appeared
(Michel et al., 2001) showing PGRP-SA to be a soluble receptor of the Toll
pathway. It was surprising that a knock-out of a soluble PGRP displayed such a
clear phenotype because the Drosophila genome comprises six very similar
38
short putatively exported PGRP proteins. The knock-out mutant placed PGRPSA upstream of Spätzle.
I felt confident that PGRP-SC1B also was a receptor that perhaps
recognized some other type of peptidoglycan and I consequently compared the
affinities of PGRP-SA and SC1B towards different peptidoglycan types.
Insoluble peptidoglycan was incubated with protein and surprisingly, the
reaction tubes with PGRP-SC1B was found to have a much smaller (or no)
peptidoglycan pellet as compared to those with PGRP-SA present. We then
recollected the relationship of PGRP to phage T7 lysozyme and its enzymatic
nature. The realization that PGRP-SC1B possesses enzymatic activity readily
explains the diminished peptidoglycan pellets. This finding was also the starting
point of a new track of PGRP research.
Enzyme characterization (Paper I)
Insoluble peptidoglycan isolated from bacteria is a complex polymeric
macromolecule of undefined size. Using peptidoglycan as an enzyme substrate
limits the possibilities to define enzyme kinetic constants that typically require a
defined substrate that is converted to a defined product possible to measure. To
obtain a rough estimate of the enzymatic properties of PGRP-SC1B we analyzed
the rate by which a turbid solution of peptidoglycan turns clear. We found that
PGRP-SC1B indeed is an efficient enzyme with an activity in the same range as
hen egg white lysozyme. The highest activity was recorded against S. aureus
peptidoglycan and we found that the presence of teichoic acids had an inhibitory
effect. PGRP-SC1B has a higher pH optimum than lysozyme and if the two
enzymes are compared at pH 8.0 instead of pH 7.2, SC1B actually shows a
higher activity against S. aureus peptidoglycan than lysozyme (unpublished
data). The crystal structure of T7 lysozyme shows a mixed α/β conformation
with a prominent cleft with a zinc ion bound to three amino acid ligands (Cheng
et al., 1994). A mutational analysis pointed out two additional residues as being
required for the amidase activity. Aligning the active-site residues of T7
lysozyme and PGRPs made evident that SC1B retains all five residues.
Interestingly, in total six Drosophila PGRPs have these residues conserved and
this is also true for the long mammalian PGRPs (PGRP-L). Amidase activity has
later been shown also for Drosophila PGRP-LB and for the mouse and human
PGRP-L (Gelius et al., 2003; Kim et al., 2003; Wang et al., 2003). Interesting to
note was that none of the receptor PGRPs has all the five residues retained. A
critical cysteine residue, being one of the zinc ligands, is replaced by a serine
residue in the peptidoglycan binding receptor-PGRPs i.e. SA, SD, LE, LCx, and
B. mori PGRP. We decided to examine what role this cysteine had for SC1B
activity. By mutagenesis I did exchange it with a serine or an alanine residue. As
expected, the cysteine was absolutely required for the enzymatic activity but the
mutants had not lost their peptidoglycan binding capabilities. To more
39
specifically determine the enzymatic specificity of PGRP-SC1B, we undertook
an analysis of the cleavage products by means of HPLC fractionation and
determination of molecular masses by Maldi-TOF mass spectrometry. The mass
spectrum shows a number of peaks but all correlate to products produced by the
action of an N-acetylmuramoyl L-alanine amidase cleavage and it was firmly
established that PGRP-SC1B has the same activity as T7 lysozyme.
As described in Paper II we have also cloned and characterized
PGRP-SB1, which is one of the amidase candidates postulated in Paper I. We
experimentally demonstrate that it is an enzyme with N-acetylmuramoyl Lalanine amidase activity and that it has a preference for DAP-type
peptidoglycan. This stands in contrast to PGRP-SC1B that has a broader activity
spectrum and are highly active against peptidoglycan of both Lys-type e.g. S.
aureus peptidoglycan, and of DAP-type e.g. E. coli peptidoglycan. The best
substrates for PGRP-SB1 are peptidoglycan from B. megaterium and E. coli.
A peptidoglycan scavenger (Paper I)
The T7 lysozyme mediates lysis of the E. coli cell to facilitate the release of
newly synthesized phage particles from the infected bacterium. To examine if
PGRP-SC1B is bacteriolytic we extensively assayed antibacterial activity
against a range of different bacteria but with negative results. What could the
physiological role of a peptidoglycan degrading enzyme be if it was not
antibacterial? As peptidoglycan is a highly immune-stimulatory macromolecule,
a possible function could be to modify this property. We speculated that the
degradation products could either give a stronger immune response than the
intact cell wall because the ligands would more widely spread to cells and
tissues, or else that the degradation products would be less stimulatory. To test
this we challenged Drosophila cells with the SC1B degradation products and it
was clear that the immune induction was much reduced as compared to intact
peptidoglycan or lysozyme-degraded peptidoglycan. This observation led us to
suggest a scavenger function for the protein. Peptidoglycan fragments will most
likely spread systemically when bacterial lysis is caused by antibacterial
peptides or other factors during an infection. A peptidoglycan scavenger
function would be beneficial for the host in order to restrict the duration of the
immune response and thereby limit the damages caused to own tissues. To bring
levels of immune stimulatory molecules back to zero is likely a prerequisite for
the fly to be able to respond to a second infection. The scavenging effect of
PGRP-SC1B is readily explained because it cleaves the same motif in the
peptidoglycan molecule that is being used by the receptor-PGRPs. It must be
stressed, however, that this is an indicative observation in vitro and a possible
PGRP scavenger function still remains to be shown in vivo. The likelihood of
PGRP-SC1B being a scavenger can be further discussed as the expression
pattern of PGRP-SC1B suggests that the main expression is restricted to the gut
40
region. However, there is also a weak expression from the fatbody (Werner et
al., 2000). Possibly, SC1B is exported to the gut and is taking part in bacterial
degradation as a digestive enzyme. This would be in analogy with the six
Drosophila lysozymes that are expressed in the gut (Daffre et al., 1994) and
might play a role in the degradation of the bacteria present in the decomposing
fruits on which larvae feed.
A bactericidal PGRP amidase (Paper II)
As discussed above, PGRP-SC1B did not show any antibacterial activity and
therefore it was surprising to find that PGRP-SB1 is highly bactericidal against
B. megaterium. However, this is the only bacterial species PGRP-SB1 showed
activity against, despite an exhaustive search for antibacterial activity against
other bacteria including several Bacillus species. At present we do not know the
feature(s) of the B. megaterium cell wall that makes the peptidoglycan
accessible for PGRP-SB1 degradation. The removal of teichoic acids enhances
the activity of PGRP-SC1B in vitro (Paper I). A possible explanation to the
PGRP-SB1 bactericidal activity could be that the distribution or composition of
teichoic acids being different in B. megaterium compared to other bacilli.
LPS – to be, or not to be, an elicitor of the Drosophila immune system
(Paper III and IV)
Bacterial lipopolysaccharide, LPS, has for long been known to
induce inflammation in humans. It was the first bacterial pattern molecule to be
linked to a specific receptor (TLR4) of the human innate immune system
(Poltorak et al., 1998; Rock et al., 1998). Also in studies of the insect immune
system, LPS has been extensively used to elicit immune gene expression. An
LPS receptor was therefore expected to be found in control of the Drosophila
immunity pathways. The reports that describe PGRP-LC as the Imd-pathway
receptor clearly show that the knock-out flies have a hampered response to LPS
and live Gram-negative bacteria in addition to peptidoglycan (Choe et al., 2002;
Gottar et al., 2002; Ramet et al., 2002b). However, PGRP proteins had been
shown to bind peptidoglycan and not LPS. Even if peptidoglycan is a constituent
of the Gram-negative bacterial cell-wall, it is hidden underneath the LPScontaining outer membrane. It is not easy to envisage how a receptor would be
able to penetrate the lipid-bilayer to get access to the peptidoglycan. The
observation that the LPS response is blocked in the PGRP-LC mutant fly
indicates a broader PGRP-LC ligand specificity. However, an alternative
explanation could be the presence of peptidoglycan impurities in the LPS
preparation. The purity issue has been central in the study of mammalian Toll41
like receptors and their ligands. TLR2, for example, was initially believed to be
an LPS receptor until extensive re-purification of LPS abolished the TLR2
mediated response (Hirschfeld et al., 2000). However, our discovery of a
peptidoglycan scavenger, PGRP-SC1B, as described in Paper I, presented a way
to decontaminate LPS from peptidoglycan impurities. In the two studies
presented in Papers III and IV, PGRP-SC1B-treated LPS has been used to
challenge Drosophila cells. In both studies this LPS is eliciting an immune
response, in Paper IV at a concentration as low as 10 ng/ ml. The conclusion
from Paper III is therefore that LPS really is an elicitor of the Imd-pathway. This
conclusion, however, was drawn under the assumption that the peptidoglycan
fragments produced by PGRP-SC1B lack activity. The study in Paper IV takes
the impurity issue a step further and analyses the LPS preparation after it has
been fractionated by gelfiltration. Indeed, the material that induces Drosophila
cells from the LPS-solution that had been treated with a peptidoglycan digesting
enzyme has a lower molecular weight than un-treated LPS. This is not the case
for the LPS-responsive HEK293/TLR4 cells, which respond to material of the
same molecular weight in the treated and un-treated LPS. Paper IV clearly
shows that Drosophila S2 cells, having a functional Imd-pathway, are
unresponsive to LPS.
The first study suggesting that LPS is not an inducer of the Imdpathway was from Bruno Lemaitre’s laboratory (Leulier et al., 2003). They
showed instead that the Toll and the Imd-pathway discriminately can be
activated by peptidoglycan of Lys-type and DAP-type, respectively. An LPSinjection to the flies gave the same response as an injection of sterile water.
However, the response from the sterile control injection was high and could
possibly have masked a weak LPS response.
PGRP-LC – three of a kind (Paper III and IV)
The PGRP-LC gene-cluster encodes membrane-bound proteins generated by
alternative splicing. A common intracellular non-PGRP domain and a
transmembrane domain are spliced together with any of three different PGRP
domains LCa, LCx, or LCy. Two additional PGRP domains was thought to be
part of the PGRP-LC gene-cluster but as shown in paper III these are transcribed
as a single transcript including both domains and therefore defines a unique
gene, PGRP-LF.
Gene knock-out experiments have placed PGRP-LC as a crucial
component of the Imd-pathway up-stream of the Imd-protein (Choe et al., 2002;
Gottar et al., 2002; Ramet et al., 2002b). In one of these studies a chromosomal
mutation in the non-PGRP domain, thus affecting all three spliceforms, totally
blocks the Imd-pathway (Gottar et al., 2002). A similar phenotype was reported
in one of the other studies characterizing a chromosomal mutation affecting the
PGRP-LCx domain (Choe et al., 2002).
42
In studies presented in Paper III and IV a similar approach was
employed to determine the relative importance of the three different splice
forms. In a cell culture system, RNAi was directed toward the different spliceforms to knock-down their expression, one at a time. Different immune elicitors
were then added and the effect on immune gene induction was assayed. The
results show that regardless of immune elicitor, RNAi directed against the
PGRP-LCx domain or against the common non-PGRP domain totally block the
Imd-pathway. This supports the notion that PGRP-LCx is a necessary spliceform and a major receptor of the Imd-pathway. RNAi directed against PGRPLCy, on the other hand, has no effect at all on Imd-pathway activation by the
stimuli used. The effect of RNAi directed against the PGRP-LCa spliceform is
intricate. In Paper III we report that LPS treated with PGRP-SC1B (but not
untreated LPS) or living Gram-negative bacterial cells is dependent on PGRPLCa. The results in Paper IV directly show that LCa is required for induction
with monomeric peptidoglycan. Also shown is that the PGRP-SC1B treatment
does not fully remove the stimulants from LPS but reduces the immunostimulatory capacity 100 times. Is it possible that only Lys-type peptidoglycan
can be fully inactivated by PGRP-SC1B? A more recent paper from our
laboratory (Gelius et al., 2003) shows that even if PGRP-SC1B is highly active
against E. coli peptidoglycan it can not cleave TCT. Thus, PGRP-SC1B can
degrade most of the E. coli peptidoglycan but leave monomeric TCT-like
molecules. As shown by Stenbak and colleagues (Stenbak et al., 2004), a
peptidoglycan monomer with a muramic acid in anhydo conformation in
combination with a DAP residue is required for full Imd-pathway activation. It
is thus possible that the immune eliciting factors giving an LCa-dependent
response in Paper III in fact are TCT-like molecules. Taking this into account
provides an explanation why similar experimental results were interpreted
differently in Paper III and IV.
Polymeric peptidoglycan recognition (Paper III, IV, and V)
It has for long been realized that the Toll pathway mainly responds towards
fungi and certain Gram-positive bacteria such as M. luteus and that the Imd
pathway is activated by Gram-negative bacteria and some Gram-positives such
as Bacillus species. It was recently suggested that the receptors of the two
pathways could discriminate between peptidoglycans having an L-lysine or a
meso-DAP residue in the branching position of the tetrapeptide (Leulier et al.,
2002).
As no biochemical data were available, we decided to examine the
relative binding affinities of the PGRP receptors and to correlate these with the
known elicitor specificities of the two pathways. As shown in Paper V PGRPSA, the soluble receptor of the Toll-pathway indeed has affinity for
peptidoglycan of Lys-type and not for peptidoglycan from B. megaterium, being
43
of DAP-type. Somewhat surprising however, we found that PGRP-SA has good
affinity to DAP-type peptidoglycans from other species (E. coli and L.
plantarum). In future experiments, it would be interesting to examine how well
these peptidoglycans activate the Toll pathway.
The peptidoglycan binding data for PGRP-LCx, the receptor of the
Imd-pathway, are more intriguing. PGRP-LCx shows a remarkable high affinity
for all types of peptidoglycan, which stands in contrast to the DAP-type
peptidoglycan preference that has been reported for the Imd-pathway activation.
A possible explanation of the strong activation seen with DAP-type
peptidoglycan could be the anhydro-MurNAc present at the reducing ends of the
glycan strands in Gram-negative bacterial peptidoglycan. The anhydo bond not
only confers a different appearance to the MurNAc residue but also fixes the
amino-hexose in a locked conformation in contrast to the open conformation of
unreduced MurNAc that is oscillating between α- and β-conformation. It is thus
possible that even if PGRP-LCx can bind to any polymeric peptidoglycan, the
presence of an anhydro-MurNAc is required for a strong induction of the Imdpathway. This idea is supported by in a recent study (Stenbak et al., 2004). They
found that the anhydro-MurNAc in combination with a DAP residue indeed is
required for a full Imd response. Another recent study also supports this idea
that the conformation of the MurNAc is instrumental for Toll and Imd activation
(Filipe et al., 2005). These investigators show that the Toll pathway, in contrast
to the Imd pathway, is positively activated by peptidoglycan fragments in which
the MurNAc has an unreduced flexible conformation.
In the model, presented in Paper V, we postulate that an Imdactivating PGRP-LCx homodimer will form when one of the dimer partners
binds to a peptidoglycan motif that includes an 1,6-anhydro MurNAc residue
and the other binds to an adjacent common peptidoglycan motif.
Monomeric peptidoglycan recognition (Paper III, IV, and V)
The analysis of the relative requirements of the different PGRP-LC spliceforms
for activation of the Imd-pathway, presented in Paper III and IV, suggests that
both the PGRP-LCa and LCx form are needed when the Imd-pathway is
activated by monomeric peptidoglycan, or by impure LPS. These results suggest
that the activating unit is a heterodimer complex formed by PGRP-LCa and LCx. In our biochemical analysis of the recombinant PGRP-LC domains,
presented in Paper V, we found unexpectedly that PGRP-LCa does not bind to
monomeric peptidoglycan and that instead PGRP-LCx has affinity for this
elicitor. As activation data could be explained by receptor dimerization, an assay
was set up that would enable testing if the proteins possibly get affinity for each
other in the presence of the proper ligand. In this assay one of the recombinant
proteins contains a histidine-tag and the other protein interaction partner lacks
such tag. The two protein forms are incubated in the presence or absence of a
44
ligand and the incubation mixture is passed through a solid-phase histidinebinding matrix. If the proteins have affinity for each other then they are both
trapped in the matrix, otherwise only the tagged form stays bound. In a second
step, the proteins that are bound to the matrix are eluted. Using this method, we
found that PGRP-LCa and -LCx indeed have affinity for each other in the
presence of monomeric peptidoglycan (TCT). The interaction is specific for
these PGRP forms since dimerization is not induced by any other combination
of PGRP-LCa, LCx and SA. Our model for Imd activation is mainly based on
the results from paper III, IV, and V. It is proposed that the Imd-pathway can be
activated by PGRP-LCx binding to monomeric peptidoglycan, which causes a
minor conformational change that attracts PGRP-LCa. The two proteins will
thus constitute an activating dimer-complex with LCa serving as an adaptor or
transducer and not as a receptor. Again, I want to stress that this model of Imdpathway activation is based on experiments in vitro using cell lines and
recombinant proteins.
Note: during the preparation of this thesis an article was published
describing the crystal structure of PGRP-LCa (Chang et al., 2005). The structure
shows that LCa has a distorted binding groove. Binding data presented are in
full consonance with, but do not add anything to, the findings we had already
published in paper V.
Concluding remarks
The work in this thesis has been focused on the recognition of bacteria by innate
immune receptors which initiate the triggering of the pathways controlling
immune gene expression. Most of the experiments have been performed in vitro
using biochemical protein interaction approaches and molecular biology
techniques to derive and manipulate cell lines.
The main goal has been to elucidate the biological role of members of
the PGRP gene family. Many questions have been answered and many new
questions have been formulated. It is still an open question how Gram-negative
bacteria are being recognized as we showed that LPS is not an inducer of the
Imd-pathway. If the peptidoglycan sacculus is being recognized then PGRP
proteins must in some way penetrate the LPS-containing outer membrane.
Alternatively, peptidoglycan monomers which are lost to the environment by
growing Gram-negative bacteria could possible offer an explanation to how
these bacteria are being recognized. The existence of the dedicated splice variant
PGRP-LCa indicates that recognition of peptidoglycan subunits is instrumental
for bacterial recognition. We have demonstrated that the heterodimerization of
PGRP-LCx and -LCa can be induced by monomeric peptidoglycan and it is
likely that homodimerisation of PGRP-LCx receptors will be a consequence of
polymeric peptidoglycan binding. However, these dimerization events remain to
be shown also in vivo.
45
The elucidation of the physiological roles played by the peptidoglycan
degrading enzymes with N-acetylmuramoyl L-alanine amidase activity will have
to await the establishment of knock-out mutants. One can, however, foresee that
they play important roles by modulating the property of the major stimulant of
the Drosophila immune system, namely peptidoglycan.
46
Acknowledgements
Hmm, have you already been reading this far? Noo, I hardly believe you…
Maybe you didn’t know that those who skip pages in this book will be reborn as
a laboratory fly in their next lives?
First, I want to express my gratitude for my supervisor professor Håkan Steiner
who brought my attention to this interesting field of pattern recognition. I
appreciate the warm and open atmosphere in your lab, the free hands you gave
me, and the immense support I had whenever I needed it. Your generosity in
sharing your knowledge (and humor) has been invaluable. -I will never forget
the mantra ‘PGRP - besser als Toll’…
Next, I want to thank the other inhabitants of our research group from past
and present; Gang for introducing me to the lab during my exam-work time,
Annika for ‘lending’ me numerous acrylamide gels, Eva for fruitful discussions
concerning amidases and for a never-ending flood of Swedish/ German jokes,
Jenny, the best collaborator ever, who will never learn to say ‘kex’, Carina for
your unpredictable comments on all aspects of life - I will be reminded of you
whenever I’ll eat any Grapefruit, Sandra for your warm attitude and for
dragging me around in the forest every week, and MacGyvor for fixing the
machines and tools that I occasionally broke.
The other seniors at the microbiology department, Leif, for introducing
me to “who is who in the bacterial community”, Monica, for recurrent updates
on male javelin throwers… Margareta for being supportive in teaching issues,
IngaBritt, IB, for chats and laughs in the kitchen, cross-word solving, massage,
and hjortron-berries, Helena, Anita, Olga, Eva, and Annette, for lunch-time
chats and for keeping the institution going, Björn for preventing my computer
from crashing, Inger Holmgren for extensive support at the teaching lab and for
all the cakes and cookies.
My comrades at the translation lab: Ernesto, Victor, Qing (how could
you possibly write your thesis that fast? I’m impressed), Sergey, Richard and
Mirja for many interesting discussions
Georgina and Lily, an advice for your own best, -keep yourselves
updated on male javelin throwers (especially those from the Czech Republic)…
The people that have left the department during the years: Peter H for one
or two games of chess, badminton and billiard, Oliver take care of Jenny and
Leon in Copenhagen, Martin for your anti-optimism, Magnus, Magda and
Josefin our parties have been missing you, Ann for all the rules and for all the
cakes, Robert S, for exiting sub-cloning discussions.
The staff at the rest of our department, the Geneticists and Toxicologists,
especially the athletes attending the Innebandy fights.
47
The other innate immunity researchers at the university from Ylva’s,
Uli’s, Ingrid’s groups, you are truly a resource of knowledge!
Reseach collaborators: Dan Hultmark and your group in Umeå for nice
collaboration, it is always inspiring to discuss with you. Niklas Schultz for
sharing your knowledge and for running the confocal, Janet Håkansson for
being an outstanding exam-worker, Neal Silverman, Takashi Kaneko, and
William Goldman for collaboration, TCT, and research discussions.
People outside the university…
Thomas and Richard for bowling and for crazy parties in the ‘good old
days’, Mathias and family for billiard games and dinners, Eddy, Helen, Emma,
Viktor, and Lovisa for numerous dinners and for helping me reinvent the
relativity-theory during late night-hours, Niklas alias Gegge for chips and
electricity know-how.
My sister Gunlis, and family, Olle, Fred and Annika for extensive
hospitality and support during one of the toughest periods of my life.
My brother Roger, and family, Anna-Karin, Emmie and Lise for care,
and for introducing me to ornithology, which was the main reason I started this
education in the first place (-how did I end up with this fly research? I still
cannot give a proper answer…).
My parents Margit and Harry, thanks for your support, it is always so
nice to come to your place and to be taken care of…
Finally, my family,
-Therese, for love, support, Grådask, and for making this work possible
-Fabian and Johannes, you will definitely be the next Zlatan, but times two!
-min dotter Stina, du är mitt käraste skogssmultron ♥ Tack för att du låter UllaBerta och Doris låna mig en arm att sova på ibland!
6
Bryt upp, bryt upp! Den nya dagen gryr.
Oändligt är vårt stora äventyr.
48
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