The systemic immune response AMPs

Among the various molecules produced by the fat body in response to infection, AMPs are the best characterized (reviewed in Imler and Bulet, 2005). Gene transcripts encoding AMPs are not detected in uninfected conditions, and molecular studies have revealed that their expression is induced upon infection. Over 20 AMPs, which comprise seven classes, have been identified. They are small (<10 kDa; with the exception of the 25 kDa Attacin), cationic, and exhibit a broad range of activities against bacteria and/or fungi (Figure 2.1). Insect AMPs are active at the microbial membrane, and while their precise mode of action is still unclear, the specificity of their activity in response to infection is well characterized. Diptericin, Drosocin, and Attacin are very effective against Gramnegative bacteria (Wicker et al., 1990; Bulet et al., 1993; Asling et al., 1995). Defensin is active against Gram-positive bacteria (Dimarcq et al., 1994), whereas Drosomycin and Metchnikowin exhibit antifungal activity (Fehlbaum et al., 1994; Levashina et al, 1995). Cecropin A1 acts against both bacteria and some fungi (Ekengren and Hultmark, 1999). Antimicrobial peptides are generally encoded by intron-less genes that are located in family clusters on the chromosome. Gene amplification by recombination and gene conversion are assumed to be the genetic forces driving the evolution of AMP genes (Sackton et al, 2007). It should be noted that Drosophila also encodes 13 lysozymes, but they do not appear to contribute to the systemic response. Rather, they play a digestive role in the gut (Hultmark, 1996). To date, no loss-of-function study has addressed the relevance of individual peptides, but indirect evidence supports their primary role in Drosophila immunity (Tzou et al., 2002; Liehl et al, 2006).

2.2.1 Regulation of AMPs by Toll and Imd pathways

Two pathways, Toll and Imd, have been shown to regulate AMP genes (Lemaitre et al, 1995, 1996). These two pathways share many common features with the mammalian Toll-like receptor (TLR) and tumour necrosis factor a (TNFa) signalling cascades, and regulate NF-kB transcription factors (Ferrandon et al., 2007). The Toll pathway is triggered by the proteolytic cleavage of the Toll ligand, the cytokine Spätzle (Spz), and leads to activation of the Rel proteins Dif and Dorsal. This pathway is activated by both Gram-positive bacteria and fungi and it controls, to a large extent, the expression of AMPs with activity against fungi (e.g. Drosomycin). In contrast, the Imd pathway mainly responds to Gram-negative bacterial infection and controls antibacterial peptide genes (e.g. Diptericin) via the activation of the Rel protein Relish. Thus, the immune system of Drosophila demonstrates how two distinct signalling pathways can modulate the expression of genes in response to different classes of microbes, and can serve as a simple model to decipher innate immune mechanisms.

2.2.1.1 Toll pathway

The Toll pathway, as illustrated in Figure 2.2, is a conserved signalling cascade that was initially identified for its role in the establishment of dorso-ventral polarity of the embryo (Belvin and Anderson, 1996). Subsequently, it was implicated in additional developmental processes and the regulation of the systemic immune response, for which its function has been well characterized. Toll is a transmembrane receptor with an ectodo-main composed of leucine-rich repeats (LRRs) and an intracellular Toll/interleukin-1 receptor (TIR) domain (Hashimoto et al., 1988). Toll is activated by dimerization upon binding with a cleaved form of the secreted protein Spätzle (Weber et al., 2003;

Septic injury

Systemic response

Antimicrobial peptide expression

Septic injury

Systemic response

Antimicrobial peptide expression

Antimicrobial Peptide

Diptericin-lacZ expression in the gut

Figure 2.1 Overview of Drosophila systemic and local antimicrobial responses. Direct injection of bacteria into the haemolymph (septic injury) elicits a systemic immune response in the fat body, while ingestion of bacteria elicits a local immune response in the gut epithelium. Both tissues secrete a range of antimicrobial peptides with different activity spectra (classed here by their principal activity). Diptericin-lacZ expression in the fat body and gut is visualized by X-gal coloration.

Diptericin-lacZ expression in the gut

Figure 2.1 Overview of Drosophila systemic and local antimicrobial responses. Direct injection of bacteria into the haemolymph (septic injury) elicits a systemic immune response in the fat body, while ingestion of bacteria elicits a local immune response in the gut epithelium. Both tissues secrete a range of antimicrobial peptides with different activity spectra (classed here by their principal activity). Diptericin-lacZ expression in the fat body and gut is visualized by X-gal coloration.

Fungi Yeast Gram-positive bacteria

Necrotic

Gram-negative bacteria

Stress/injury

Lysine-type peptidoglycan

Dap-type peptidoglycan

Haemocyte

GNBP3

Persephone

GNBP1

Monomeric peptidoglycan

PGRP-SA

Pro-Spätzle Spätzle

PGRP-SA

Pro-Spätzle Spätzle

Haemolymph

Jak Stat Epithelium

DD DED

dFADD

Dredd

Cytoplasm Nucleus

Dorsal Dif

Monomeric peptidoglycan

Polymeric peptidoglycan

Stress/injury

Haemocyte

</ Cytokines

PGRP-LE PGRP-LCx PGRP-LCa

PGRP-LE

</ Cytokines

Haemolymph dFADD

Dredd

DD DED

PGRP-LE

PGRP-LE PGRP-LCx PGRP-LCa

Domeless

DIAP2

Hopscotch

Domeless

DIAP2

Cytoplasm Nucleus

IRD5

Hopscotch

(JAK) (TyrKc'SH^SffiVyrKc

SH2 Y SH2

Dorsal Dif

Antimicrobial response e.g. Diptericin

Kenny

SH2 Y SH2

Kenny

Stat92E (STAT)

Antimicrobial response e.g. Drosomycin Figure 2.2 Continued

Antimicrobial response e.g. Diptericin

Stress/injury response e.g. Turandot, Tep2

Hu et al., 2004). Upon activation, Toll recruits a set of TIR- and/or death domain-containing adaptors that lead to activation of the kinase Pelle. Pelle, by an as yet unknown mechanism, leads to proteosomal degradation of Cactus, an inhibitor that maintains the cytoplasmic localization of two transactivators of the NF-kB family, Dif and Dorsal. The nuclear translocation of Dorsal and Dif induces the expression of many immune genes via binding to kB DNA motifs found in their promoters (Ip et al, 1993; Engstrom et al, 1993; Kappler et al., 1993; Busse et al., 2007). Flies carrying loss-of-function mutations in all components of the Toll pathway, except Cactus, are viable but extremely sensitive to infection by Gram-positive bacteria and fungi (Lemaitre et al., 1996; Rutschmann et al., 2002; Tauszig-Delamasure et al., 2002). Importantly, these Toll-deficient flies do not exhibit proper expression of Toll-dependent proteins and peptides, such as the antifungal peptide Drosomycin.

The extracellular steps leading to the activation of Toll by its ligand Spätzle, share similarities with the coagulation proteolytic cascades of mammals or the melanization reaction of arthropods (Krem and Cera, 2002). Infection of the host by Gram-positive bacteria or fungi activates the proteolytic activity of clip-domain serine proteases (Piao et al, 2005). This allows an amplification of the signal ending in the processing of the cytokine Spätzle by the terminal serine protease called Spätzle-processing enzyme (SPE) (Jang et al., 2006). Three distinct cascades of serine proteases are activated by different classes of microbes functioning upstream of SPE. To date, these cascades are still poorly characterized in Drosophila. An in vitro study in another model insect, Tenebrio molitor, in which the entire cascade was reconstructed with purified proteases, suggests that it functions in three steps, with each active form cleaving and activating a downstream protease (Kim et al., 2008; also see Chapter 3 in this volume). Direct recognition of the micro-organism by PRRs activates two of these cascades, whereas the third cascade might be activated by the pro-teolytic activity of virulence factors secreted by the pathogen (Gottar et al., 2006). These cascades require tight regulation to avoid the potential of aberrant activation. To this end, serine protease inhibitors of the Serpin family, such as the serpin Necrotic, provide multiple layers of control to maintain proper activation of the Toll pathway (Levashina et al., 1999). In this manner, regulation of the humoral response by the Toll pathway requires

Figure 2.2 Principal pathways regulating the systemic response in Drosophila (reviewed in Agaisse and Perrimon, 2004; Ferrandon et al., 2007; Lemaitre and Hoffmann, 2007). Three distinct pathways regulate the response to microbial infection: the Toll pathway (activated mainly by fungi and Gram-positive bacteria), the Imd pathway (activated mainly by Gram-negative bacteria), and the JAK/STAT pathway activated by stress/injury sensed by haemocytes. Toll pathway (left): the Toll receptor is activated upon binding a cleaved form of Spätzle. Proteolytic cascades initiated by secreted recognition molecules (PGRP-SA and Gram-negative-bacteria-binding protein 1 (GNBP1) for Gram-positive bacteria, GNBP3 for ß-glucans) or by direct cleavage of serine proteases (by fungal virulence factors) converge on Spätzle processing enzyme (SPE), which cleaves Spätzle. Spätzle binding induces Toll dimerization and subsequent recruitment of MyD88, Tube, and Pelle, leading to phosphorylation and proteasomal degradation of Cactus. Cactus degradation allows the Rel transcription factors Dif and Dorsal to translocate to the nucleus where they bind NF-KB-response elements and activate transcription of genes including Drosomycin. Imd pathway (centre): Imd is recruited by PGRP-LC upon direct binding to monomeric (LCx/LCa heterodimer) or polymeric (LCx heterodimer) diaminopimelic acid (DAP)-type peptidoglycan. Imd then recruits dFADD and the caspase Dredd, which might be responsible for the cleavage of phosphorylated Relish. This phosphorylation is thought to be mediated by an inhibitory kB (IkB) kinase complex (IRD5 and Kenny), itself activated by TAK1. TAK1 activation of the IkB kinase is dependent on its adaptor TAB2 and Imd and possibly dFADD and DIAP2. The precise nature of these relationships remains unclear. Phosphorylation and cleavage of its ANKyrin repeats allows the Rel domain of Relish to translocate to the nucleus where it binds different NF-KB-response elements and activates transcription of genes including diptericin. JAK/ STAT pathway (right): JAK (Hopscotch), pre-associated with dimers of the receptor Domeless, is activated upon receptor binding of the cytokine Upd-3. Upd-3 itself is secreted into the haemolymph by haemocytes at sites of septic injury. JAK activation leads to recruitment of STATs (Stat92E), which are phosphorylated and dimerize, translocating to the nucleus to activate transcription of targets including Turandot genes. The stress/injury response may also be regulated by the c-Jun N-terminal kinase (JNK) pathway, which is activated by TAK1 downstream of Imd. ANK, ANKyrin repeats; BIR, baculovirus inhibitor of apoptosis repeat; CAS, caspase domain; CBM, cytokine-binding module; DD, death domain; DED, death-effector domain; FN3, fibronectin type III-like repeats; Kc, kinase domain; P, phosphate group; PGRP, peptidoglycan-recognition protein; Rel, Rel homology domain; RING, RING-finger domain; SH2, Src homology 2 domain; TIR, Toll/interleukin-1 receptor domain; TyrKc, tyrosine kinase domain.

the coordination of two building blocks, an extracellular cascade of proteases linked to PRRs and an intracellular NF-kB transactivator, that are coupled by Spätzle. Although Toll and its vertebrate counterparts, the TLRs, both play important roles in the host innate immune response, there is one major distinction between insect and vertebrate Toll functions; Toll is activated by the endogenous ligand Spatzle, while TLRs bind directly to micro-bial products. This has made it difficult to ascribe a common origin of immune function to the Toll receptors, despite their apparent similarities (Leulier and Lemaitre, 2008).

2.21.2 Imd

The Imd pathway is activated in response to Gram-negative bacteria and regulates a large set of antibacterial peptides (Figure 2.2). Mutations in genes encoding prototypic components of this pathway are fully viable, but highly susceptible to infection by Gram-negative bacteria (Lemaitre et al., 1995). This increased susceptibility is associated with the inability to activate the transcription of AMP genes. The organization of the Imd pathway differs from the Toll pathway in that it is activated by a membrane-bound PRR, peptidoglycan-recognition protein (PGRP)-LC, expressed by fat body cells (Choe et al., 2002; Gottar et al., 2002; Rämet et al., 2002). Upon Gramnegative infection, PGRP-LC recruits the Imd protein which then interacts with the adaptor dFADD via a death domain interaction (Leulier et al, 2002; Naitza et al, 2002). It is thought that dFADD then recruits the caspase Dredd (Leulier et al., 2000). Dredd is proposed to associate with Relish (Hedengren et al, 1999; Stöven et al, 2000). Following Relish cleavage, the Rel transactivator domain translocates to the nucleus, whereas the inhibitory domain remains stable in the cytoplasm. Relish is phosphorylated by the inhibitory kB kinase (IKK) signalling complex (Rutschmann et al., 2000; Silverman et al., 2000; Lu et al., 2001), which is itself thought to be activated by TAK1 and its adaptor TAB2 in an Imd- and possibly dFADD-dependent manner (Vidal et al., 2001; Gesellchen et al., 2005; Kleino et al., 2005; Zhuang et al., 2006). The Ring domain protein DIAP2 may participate via activation of dTAK1 (Gesellchen et al, 2005; Kleino et al, 2005; Leulier et al, 2006). Although the Imd pathway is well characterized from a genetic point of view, the molecular steps that couple PGRP-LC dimerization at the cell surface to the nuclear translocation of Relish remain poorly characterized.

2.2.2 Synergistic interaction between Toll and Imd pathways

Toll and Imd form two distinct pathways that can be activated independently (Lemaitre et al., 1996; Georgel et al, 2001; De Gregorio et al, 2002). However, both pathways usually function in synergy. Injection of microbes directly into the haemocoel generally leads to an activation of both pathways, although at different levels corresponding to the type of micro-organism injected (Lemaitre et al, 1997). The most important mechanism of integration of the two pathways occurs at the gene-promoter level via the presence of different Toll and/or Imd KB-responsive elements (Busse et al, 2007; Tanji et al, 2007). In addition, many components of these pathways are regulated at the transcriptional level, which provides an additional layer of regulation that both amplifies the immune response and fosters cross-talk between the two pathways (De Gregorio et al, 2002). Thus, a mutation that constitutively activates the Toll pathway results in faster activation of the Imd pathway upon subsequent infection of Gram-negative bacteria (Lemaitre et al., 1996).

Recent large-scale analyses, at the transcriptome and proteome levels, have revealed that in addition to AMPs, the production of many peptides and proteins is activated following septic injury (Figure 2.3) (De Gregorio et al., 2001; Irving et al., 2001). Further genetic evidence demonstrated the Toll and Imd pathways to be the major regulators of this response (De Gregorio et al., 2002). Some of these target genes are involved in the regulation of the systemic immune response itself (e.g., signalling components). Others participate in distinct defence mechanisms as components of the melani-zation cascade or clotting system, or opsonins. A third group of proteins includes putative immune effectors. Among this group are 17 members of the Drosophila immune molecule (DIM) family

Recognition

PGRP-LB PGRP-LC PGRP-LF PGRP-SA PGRP-SB1 PGRP-SC2 PGRP-SD GNBP-like GNBP-like 3 Idgfl ^ Idgf3 j

' Other ^ responses

Stress Turandot Frost

Iron metabolism Zip3 Transferrin

ROS metabolism Peroxidase (IRC)

Enzymes 3 Carboxylesterases

P-Galactosidase . Lipl j

Signalling

Imd

Toll

JNK

IMD

Necrotic

Pelle

d-Jun

Relish

spirit

Cactus

Puckered

Pirk

SPE

Dorsal

Spätzle

Dif

Toll

Small peptides

Known antimicrobials 4 Attacins 4 Cecropins

Defensin 2 Diptericins Drosocin 2 Drosomycins Metchnikowin

Peptides of unknown function

DIM1 DIM2 DIM4 DIM23 13 other DIMs* 21 other peptides,/

Humoral responses

Melanisation Coagulation

Pale MP1

Punch MP2

Dhpr Serpin27A

Ddc Serpin28D

yellowf laccase-like Cp19

Fondue

Opsonisation Tep2 Tep4 a2M-receptor-like

Unknown function 3 Serpins 14 Serine proteases i

Figure 2.3 Drosophila immune-regulated genes. Microarray analysis has identified the repertoire of genes regulated upon septic injury with a mixture of Gram-positive and Gram-negative bacteria or natural fungal infection (De Gregorio et al., 2001; Irving et al., 2001). Only upregulated genes are shown, organised by their putative or determined functions. * Drosophila immune molecules (DIMs ) were identified as peptides induced by the immune response, rather than upregulated transcripts (Uttenweiler-Joseph et al., 1998).

and eight Turandot proteins, which are small pep-tides secreted by the fat body with a possible role in the host stress response (Uttenweiler-Joseph et al., 1998; Ekengren et al., 2001). Furthermore, one catalase gene, two transferrin genes, and one iron-transporter gene are also induced following septic injury, suggesting a role for reactive oxygen species (ROS) and iron sequestration to limit microbial development (De Gregorio et al., 2001; Irving et al., 2001). Iron is essential for most invading microorganisms during the course of an infection, and both animals and plants have evolved elaborate immune strategies to limit iron availability to micro-organisms. In addition, many uncharacter-ized genes are upregulated upon infection. A rapid survey of target genes of the Toll and Imd pathways suggests that the Toll pathway has a predominant role in providing the secreted immune components that function in the haemolymph, such as clotting and melanization factors and has a specific antimicrobial role against invading fungi. In contrast, the Imd pathway appears to orchestrate the antibacterial responses via the regulation of most antibacterial peptides. Accordingly, the Imd pathway is activated with more rapid kinetics, peaking at 6 h, while Toll regulates many late responsive genes (24-48 h). Despite our knowledge of the target genes of each pathway, their specific contribution to host defence is not completely understood and remains a major challenge in the field.

2.2.3 Two pathways, specific microbial elicitors

The initial observation that different classes of micro-organisms induced specific patterns of AMP expression, implies that Drosophila is able to sense and discriminate between microbes (Lemaitre et al., 1997). The basis of this specificity is through host recognition of microbial molecules by proteins called PRRs. These receptors interact directly with conserved motifs of micro-organisms referred to as pathogen-associated molecular patterns (PAMPs) (Medzhitov and Janeway, 1997). In Drosophila, recognition of bacteria is achieved largely through the sensing of specific forms of peptidoglycan by PGRPs (Figure 2.4) (reviewed in Steiner, 2004; Royet and Dziarski, 2007). Peptidoglycan is an essential component of the cell wall of both Gram-negative and Gram-positive bacteria. It consists of long gly-can chains of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) residues that are cross-linked to each other by short peptide bridges. Peptidoglycan is a highly complex and fast-evolving molecule with marked differences from one bacterium to another. However,

Peptidoglycan

GlcNAc ch3conh

MurNAc ch3ch ho -o.

GlcNAc ch2conh

MurNAc

Peptide bridge ch-ch3 ch3conh co l-Ala

d-Glu

d-Ala

meso-DAP d-Ala

MurNAc GlcNAc d-Ala

meso-DAP I

co l-Ala

d-Glu

meso-DAP

d-Ala conh oh d-Glu

co l-Ala ch-ch, o o^rr ch,oh/or ch3conh MurNAc GlcNAc

ch oh o

Peptidoglycan-recognition proteins ED

PGRP-SB1

PGRP-LA I I

PGRP-LC I I

PGRP-LD I I

PGRP-LE

PGRP-LF I I

PGRP-SA

PGRP-SD

KEY * * **C

* * **x Ii iM

1 III

1 1

Catalytic Signal

Membrane

Non-catalytic

PGRP domain peptide

domain

PGRP domain

Peptidoglycan binding by PGRP-LE

PGRP-LE

Peptidoglycan binding by PGRP-LE

PGRP-LE

Center Dap Peptidoglycan
TCT

Figure 2.4 Peptidoglycans and PGRPs. Recognition of bacteria is mediated by the detection of peptidoglycan, a critical bacterial cell wall component. Peptidoglycan is a peptide cross-linked polymer of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Depicted is the structure of Escherichia coli peptidoglycan, which, as in all Gram-negative bacteria, contains a meso-DAP residue in the peptide bridge, where most Gram-positive bacteria have an L-lysine. The terminal peptidoglycan monomer (highlighted) of all E. coli peptidoglycan glycan chains contains a unique variant of MurNAc with an internal 1,6-anhydro bond. This monomer is the minimal signature for detection of peptidoglycan and is referred to as tracheal cytotoxin (TCT). PGRPs bind peptidoglycan by a conserved 160 amino acid PGRP domain (related to bacteriophage T7 lysozyme). The Drosophila genome contains 13 PGRP genes, divided into Short and Long classes by their transcript length and the presence or absence, respectively, of a signal peptide. Catalytic or amidase PGRPs conserve zinc-binding residues (*), including a diagnostic C-terminal cysteine, required for the amidase activity which cleaves the peptidoglycan peptide bridge from the sugar backbone (dashed line on the top peptidoglycan molecules). PGRPs lacking these residues are referred to as recognition PGRPs. Illustrated is the three-dimensional structure of PGRP-LE binding to TCT (Kaneko et al., 2006).

peptidoglycan from Gram-negative bacteria differs from most Gram-positive peptidoglycan by the replacement of lysine with meso-diaminopimelic acid (meso-DAP) at the third position in the pep-tide chain. There is, however, a subclass of Grampositive bacteria including Bacillus species, which possess DAP-type peptidoglycan. Additionally, the localization of peptidoglycan in the cell wall is different between Gram-negative and Gram-positive bacteria. Gram-negative peptidoglycan consists of a single layer and is hidden in the periplasmic space underneath the outer membrane and lipopolysac-charide layer, whereas peptidoglycan from Grampositive bacteria is multilayered and exposed at the bacterial surface. Studies using highly purified bacterial compounds have shown that the specificity of Imd to Gram-negative bacteria is a result of receptor activation by DAP-type peptidoglycan, whereas the Toll pathway is activated by Lys-type peptidoglycan (Leulier et al, 2003). In contrast to vertebrates, lipopolysaccharide endotoxin, the major component of the Gram-negative cell envelope, has no effect on Toll and Imd pathway activity, and previous results implying an interaction are explained by the presence of peptidoglycan contaminants in commercial lipopolysaccharide preparations (Leulier et al., 2003; Kaneko et al., 2004). More in-depth analysis of the Imd-dependent PGRP response to DAP-type peptidoglycan has demonstrated that both polymeric and monomeric forms are capable of activating the Imd pathway. Specifically, the GlcNAc-MurNAc (anhydro)-L-Ala-y-D-Glu-meso-DAP-D-Ala monomer, also known as tracheal cytotoxin (TCT), was identified as the minimal peptidoglycan motif capable of efficient induction of the Imd pathway (Kaneko et al., 2004; Stenbak et al., 2004). TCT provides an ideal 'signature' for Gram-negative bacteria, as this muropep-tide is positioned at the end of the peptidoglycan strand and is released from peptidoglycan (as reviewed by Cloud-Hansen et al, 2006). In contrast, the minimum structure needed to activate the Toll pathway is a muropeptide dimer of Lys-type pep-tidoglycan (Filipe et al, 2005; Kim et al, 2008).

PGRPs are highly conserved from insects to mammals and share a 160 amino acid domain (the PGRP domain) with similarities to bacteriophage T7

lysozyme, a zinc-dependent N-acetylmuramoyl-L-alanine amidase (Yoshida et al, 1996; Kang et al., 1998; Royet and Dziarski, 2007). Sequence analysis of the 13 Drosophila PGRPs points to the existence of two subgroups with either recognition or enzymic properties (Figure 2.4). Members of the first group lack zinc-binding residues required for amidase activity, but still retain the ability to bind and recognize peptidoglycan. Most of them function as PRRs. Genetic and molecular studies have shown that PGRP-LC (where L stands for long form) is the receptor of the Imd pathway involved in the sensing of DAP-type peptidoglycan (Choe et al, 2002; Gottar et al, 2002; Rämet et al, 2002). Importantly, the PGRP-LC locus encodes three iso-forms that all differ by their external PGRP domain (Figure 2.4). Cell culture assays have shown that polymeric DAP-type peptidoglycan is sensed by a homodimer of PGRP-LCx, while TCT is recognized by a heterodimer of PGRP-LCx with PGRP-LCa, the latter functioning as an adaptor (Kaneko et al, 2004). PGRP-LE encodes a PGRP with affinity to DAP-type peptidoglycan and is expressed both extra- and intracellularly (Takehana et al., 2004). A fragment of PGRP-LE corresponding to the PGRP domain alone functions extracellularly to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface (Kaneko et al., 2006). A full-length form of PGRP-LE is also present in the cytoplasm and acts as an intracellular receptor for monomeric peptidoglycan that can activate AMP expression without the requirement for PGRP-LC (Kaneko et al., 2006). Although not part of the systemic antimicrobial response, it should be noted that this full-length form of PGRP-LE also appears to be essential in haemocytes to prevent intracellular growth of Listeria monocytogenes through its ability to induce autophagy. The discovery that PGRP-LE functions as an intracellular sensor of DAP-type peptidoglycan, indicates the presence of specific immune defences of Drosophila to intracellular bacteria (Yano et al., 2008).

The Toll pathway is activated by Gram-positive bacteria via the secreted PRR PGRP-SA (where S stands for short form) in interaction with Gram-negative-bacteria-binding protein (GNBP)1 (Michel et al., 2001; Gobert et al., 2003). The exact role of GNBP1 is still a matter of debate. It has been proposed that GNBP1 cleaves peptidoglycan to promote its recognition by PGRP-SA (Filipe et al, 2005; Wang et al., 2006), while studies in other insects suggest that this protein links PGRP-SA to a downstream serine protease (Park et al, 2007).

The tertiary structures of four PGRPs have now been solved (Kim et al, 2003; Chang et al, 2004, 2005, 2006; Reiser et al, 2004; Lim et al, 2006). A prominent feature is the presence of an extended surface groove in the PGRP domain, which includes a zinc-finger cage in the catalytic PGRP-LB. The structures of PGRP-LE and PGRP-LCa/x in complex with TCT show an interaction between the peptide stem of peptidoglycan and the PGRP groove (Lim et al., 2006; Chang et al, 2006). In contrast to other PGRPs, the PGRP domain of PGRP-LCa does not possess a typical peptidoglycan docking groove, in agreement with its role as a co-receptor sensing monomeric DAP-type peptidoglycan (Chang et al., 2005; Mellroth et al, 2005).

2.2.3.2 Gram-negative-bacteria-binding proteins (GNBPs)

GNBPs form an important family of insect PRRs that contain both a glucan-binding site and a mutated glucanase domain (Lee et al., 1996). There are three GNBPs in Drosophila and one of them, GNBP1, participates in the sensing of Lys-type pep-tidoglycan (Figure 2.2). In contrast, genetic studies indicate that GNBP3 is a PRR acting upstream of the Toll pathway in the sensing of glucans derived from fungi (Gottar et al, 2006). The serine protease cascade that links GNBP3 to SPE has not yet been deciphered.

2.2.4 Sensing of virulence factors and endogenous stress signals

The activation of the Toll and Imd pathways by PGRP and GNBP recognition of microbial elicitors supports the concept of PRRs originally proposed by C. Janeway (Janeway, 1989). Current research in the field is aimed at understanding how bacteria or fungi are detected during the natural course of infection and how different microbial elicitors reach their specific PRR. Another important question is the existence of other modes of recognition that do not involve PRRs. An alternative mode of sensing is based on direct sensing of virulence factors, in a manner analogous to the guard system of plants. This mechanism appears to be central to the sensing of entomopathogenic fungi such as Beauveria bassiana and Metharizium anisopliae (Gottar et al, 2006). Spores from these fungi have the capacity to germinate on the fly and produce hyphae that can penetrate the cuticle of insects. This direct mode of entry is mediated through the abundant production of proteases, lipases, and chitinases by the fungus. It has been proposed that the presence of Beauveria bassiana is detected, independent of GNBP3, through direct activation of the Toll pathway by a fungal protease PR1. PR1 would cleave the host serine protease, Persephone, which leads to Toll-pathway activation (Figure 2.2) (Gottar et al, 2006). Thus, entomopathogenic fungi would be recognized by the presence of proteases produced in order to enter the insect.

It is important to note that sterile injury, in the absence of micro-organisms, also weakly activates the Imd and Toll pathways. It is not yet clear whether this activation is due to the presence of microbial products or to the detection of host molecules released at the wound site. However, the existence of endogenous ligands is supported by the observation that larvae with melanotic tumours induce significant AMP gene expression (Ligoxygakis et al., 2002; Scherfer et al., 2006). This suggests a possible link between melanization and systemic expression of AMP.

2.2.5 Adjusting the immune response

Extensive activation of the immune response is generally considered deleterious for the host in terms of both resource allocation and potential damage to host tissues. Indeed, aberrant activation of the Imd or the Toll pathways results in lethality (Georgel et al., 2001). Recent studies in Drosophila have revealed that multiple levels of regulation are employed to suppress Imd pathway activity and prevent excessive or prolonged immune activation (Figure 2.5).

2.2.5.1 Catalytic downregulators of the Imd pathway In contrast to recognition PGRPs, proteins referred to as catalytic PGRPs have demonstrated (PGRP-SC1A/B, -LB, -SB1/2) or predicted (PGRP-SC2)

PGRP-SC1/2

PGRP-LB

I ^ peptidoglycan

PGRP-LB

Non-immunogenic fragments V

I ^ peptidoglycan

PGRP-LF o ! PGRP-LCx

Haemolymph

Cytoplasm

Caspar

Nucleus

Transcriptional response

Figure 2.5 Negative regulation of Imd signalling. Prolonged or constitutive activity of the Imd pathway is likely to be deleterious for Drosophila, so various mechanisms have evolved to limit activation and downregulate it following an immune challenge. Detection of small quantities of DAP-type peptidoglycan is prevented by the amidases PGRP-SC1/2, which cleave peptidoglycan into non-immunogenic fragments (Bischoff et al., 2006; Zaidman-Remy et al., 2006), and PGRP-LF, which binds DAP-type peptidoglycan and may sequester it, preventing Imd activation (Maillet et al., 2008). Intracellularly, Caspar, a homologue of human FAF1, blocks the cleavage and nuclear translocation of Relish (Kim et al., 2006). Immune activation of the Imd pathway leads to upregulated expression of the amidase PRGP-LB, which cleaves peptidoglycan, and a novel protein, Pirk, which binds to PGRP-LC (Aggarwal et al., 2008; Lhocine et al., 2008; Kleino et al., 2008). These proteins downregulate the Imd pathway, limiting the duration of the immune response.

zinc-dependent amidase activity that removes peptides from the glycan chains, thereby eliminating the immuno-stimulatory activity of peptidoglycan. Secreted amidase PGRPs, such as PGRP-SC1A and PGRP-LB, scavenge extracellular peptidoglycan and prevent its binding to PGRP-LC (Bischoff et al., 2006; Zaidman-Remy et al., 2006). Interestingly, PGRP-LB is a target of the Imd pathway, establishing a negative-feedback mechanism capable of adjusting the level of the immune response to the severity of infection.

2.2.5.2 Non-catalytic downregulators of the Imd pathway

Scavenging of immune-activating peptidoglycan by amidases is only one approach used by Drosophila to downregulate Imd pathway activation. Recently, PGRP-LF, a membrane-bound non-catalytic PGRP with two PGRP domains, was demonstrated to be a key negative regulator of PGRP-LC signalling (Maillet et al, 2008). Specifically, PGRP-LF prevents PGRP-LC activation in the absence of infection, but in contrast to PGRP-SC and -LB does not affect the Imd pathway after immune challenge. The mechanism of action of PGRP-LF is not known, but may prevent aberrant activation of both the Imd and c-Jun N-terminal kinase (JNK) pathways by residual peptidoglycan fragments ingested with food or released by indigenous microbes. Additionally, Drosophila deficient in PGRP-LF exhibit defects (abnormal wings) associated with the constitutive activation of these pathways in developmental tissues such as imaginal discs. Pirk (Poor Imd response upon knock-in) a protein interacting with PGRP-LC and regulated by the Imd pathway, has been shown to regulate the Imd pathway receptor and thus, participate in the precise control of Imd pathway induction (Aggarwal et al., 2008; Kleino et al, 2008; Lhocine et al, 2008).

2.2.6 Relevance of Toll and Imd pathway to host defence

Mutations affecting the Toll and Imd pathways have their greatest impact on resistance to systemic infections by septic injury, and death is always associated with excessive bacterial or fungal proliferation (Lemaitre et al., 1996; Rutschmann et al., 2002). For this reason, it has been proposed that the Toll- and Imd-mediated systemic response plays an important role against opportunist infections that can occur upon host injury (Hultmark, 2003). Nevertheless, flies carrying mutations affecting the Toll pathway are more susceptible to natural infection with spores of Beauveria bassiana (Lemaitre et al., 1997). This demonstrates a clear role of the Toll-mediated systemic immune response for resistance against entomopathogenic fungi. To date, there is no evidence demonstrating a role of the Imd pathway in the fat body during bacterial natural infection, although this pathway has been shown to be critical for local defence against ingested bacteria in the gut (Liehl et al., 2006; Ryu et al,, 2006; Nehme et al, 2007).

2.2.7 Contribution of additional pathways to Drosophila immune response

While Toll and Imd are the main regulators of the Drosophila immune response, additional pathways have emerged as important participants in the systemic immune response (Figure 2.2). Gene expression profiling has identified a subset of Drosophila immune-response genes that are regulated by the JAK/STAT pathway, namely genes encoding the complement-like protein TEP2 and Turandot stress proteins (Agaisse et al, 2003). It has been proposed that upon tissue damage haemocytes release a cytokine, Unpaired-3, that activates Domeless, the receptor of the JAK/STAT pathway, in the fat body. This pathway does not regulate AMPs during the systemic response but has been associated with the response to stress and tissue damage (Pastor-Pareja et al, 2008).

The JNK pathway regulates many cellular processes in Drosophila and is required for proper healing of the epidermis following injury (Rämet et al., 2001). In cell culture, JNK-dependent immune genes encode many proteins involved in cytoskel-eton remodeling (Boutros et al., 2002). Imd activates the JNK pathway through TAK1 that is thought to phosphorylate the JNK kinase basket (Silverman et al., 2003). Additionally, some negative feedbacks between the Imd-Relish and Imd-JNK branches have been reported (Park et al, 2004; Kim et al., 2005). The exact contribution of the JNK pathway to the host defence is still a matter of debate, but it has been suggested that JNK is required for AMP gene expression by the fat body (Kallio et al., 2005; Delaney et al., 2006).

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