Drosophila rely primarily on innate immune responses to effectively combat a wide array of microbial pathogens. The hallmark of the Drosophila humoral immune response is the rapid production of AMPs (antimicrobial peptides) by the fat body, the insect homologue of the mammalian liver. Production of these AMPs is controlled at the level of transcription by two NF-κB (nuclear factor κB) signalling pathways. The Toll pathway is activated by fungal and many Gram-positive bacterial microbes, whereas the IMD (immune deficiency) pathway responds to Gram-negative bacteria and certain Gram-positive bacilli. In the present review, we discuss the mechanisms involved in bacterial recognition, in particular the differential recognition of various types of bacterial PGN (peptidoglycan) by different members of the PGRP (PGN recognition protein) family of receptors.
- antimicrobial peptide
- Gram-negative bacterium
- Gram-positive bacterium
- innate immune response
- Toll pathway
PGN (peptidoglycan) is the major structural component of the cell wall of almost all bacterial species. PGN is a large, repetitive macromolecule that forms the rigid cell wall sacculus of bacteria. The basic unit of PGN is a disaccharide (GlcNAc-MurNAc) with short peptides covalently attached to the lactyl group of MurNAc. These stem-peptides include both L- and D-amino acids and are not generated by the ribosomes, but are instead synthesized by specialized enzymes that are often targets of antibiotics. The stem-peptides can also be cross-linked to one another by direct peptide bonds or short cross-linking peptides . The enzymes responsible for synthesizing PGN are excellent targets for antibiotics, and PGN itself is a potent activator of innate immune responses.
As bacterial pathogens, but not eukaryotic hosts, produce large amounts of PGN, this microbial product fits the main criteria suggested by Janeway , in his seminal paper, that innate immune elicitors are likely to be compounds unique to pathogens. In fact, the stimulation of immune responses by PGN-derived compounds was discovered more than 30 years ago, in the analysis of the adjuvanticity of mycobacterial cell wall products. MDP (muramyl-dipeptide), consisting of muramic acid-L-alanine-iso-γ-glutamate, was the first minimal PGN fragment identified based on its capacity to stimulate an immune response. More recently, several PGN-derived products have been linked to the activation of innate immune responses in mammals and insects.
Insects, such as the fruitfly Drosophila melanogaster, lack a classical adaptive immune system and rely primarily on an innate immune response to combat microbial pathogens. Even without the aid of antibodies and T-cells, the insect immune response is highly effective and is able to withstand infections with many different types of microbes. One primary mechanism that insects use to control microbes is the inducible production of a battery of AMPs (antimicrobial peptides). AMP production is controlled primarily at the level of transcription. Following infection, AMP gene expression is rapidly induced . In a pair of seminal papers, Lemaitre et al. [4,5] demonstrated that Drosophila use two distinct pathways to recognize bacterial pathogens and induce expression of AMP genes. The IMD (immune deficiency) pathway responds to all Gram-negative bacteria, whereas the Toll pathway (Figure 1) preferentially recognizes many Gram-positive bacteria . Subsequent studies, from several groups, have demonstrated that both the IMD and Toll pathways are stimulated by PGN, but differences in PGN structure determine which pathway is activated. PGN containing DAP (diaminopimelic acid) at the third position of the stem-peptide, which is found in all Gram-negative bacteria and certain Gram-positives (such as Bacillus spp. and Listeria monocytogenes), activates the IMD pathway, while lysine-type PGN, found in most other Gram-positive bacteria, stimulates the Toll pathway [7,8]. Surprisingly, lipopolysaccharide, from the outer membrane of Gram-negative bacteria, does not activate the IMD pathway although it is a potent activator of mammalian innate immunity .
PGN recognition in the Drosophila is mediated by the PGRP (PGN recognition protein) family of receptors. Drosophila have 13 PGRP genes which encode approx. 17 PGRP proteins, through alternative splicing. The PGRP domain is similar in structure to type 2 bacteriophage amidases (N-acetylmuramoyl-L-alanine amidases), enzymes that cleave the amide bond connecting the stem-peptide to the carbohydrate backbone of PGN. In fact, some PGRPs have amidase activity and are capable of digesting PGN, while others lack a critical cysteine residue that is part of the catalytic triad, and function instead as PGN-binding receptors . The Drosophila PGRPs can also be categorized based on their size. The short-form proteins, PGRP-SA, -SB, -SC and -SD, are small secreted proteins consisting of the PGRP domain plus a signal sequence, while the long-form proteins, PGRP-LA, -LB, -LC, -LD and -LE, contain additional regions. Some of the long-form PGRPs include transmembrane domains (PGRP-LA, LC and LD), while others are predicted to be cytosolic . Two non-catalytic short-form PGRPs, PGRP-SA and PGRP-SD, are involved in the recognition of lysine-containing PGN and the activation of the Toll pathway. Two long-form receptors, PGRP-LC and PGRP-LE, are necessary for the recognition of DAP-type PGN and the activation of the IMD pathway.
One well-studied agonist of the IMD pathway is a monomeric disaccharide tetrapeptide fragment of DAP-type PGN known as TCT (tracheal cytotoxin; GlcNAc-1,6-anhydro-MurNAc-L-Ala-γ-D-Glu-meso-DAP-D-Ala). All Gram-negative bacteria produce TCT during remodelling of the cell wall in the course of active growth, but most species efficiently recycle TCT by importing into the cytosol. On the other hand, some bacteria, most notably Bordetella pertussis, fail to recycle TCT and, instead, release large quantities of this monomeric PGN. TCT released by B. pertussis is cytopathic to the ciliated cells of the trachea and is believed to play a major role in causing the ‘whooping’ cough characteristic of this infection . In Drosophila, TCT is recognized by two alternative receptors. The cell-surface receptor PGRP-LC is able to directly bind extracellular TCT, while PGRP-LE detects intracellular TCT. As a cell-surface receptor, PGRP-LC is able to recognize small monomeric PGNs, like TCT, as well as larger polymeric PGN, as isolated from Escherichia coli. On the other hand, live E. coli and E. coli-derived PGN elicits little or no response through PGRP-LE, presumably because these larger polymeric PGNs cannot access PGRP-LE within the cytosol. PGRP-LE is sufficient to recognize TCT injected into fruitflies . These results suggest that Drosophila cells have mechanisms to import small fragments of PGN into the cytosol for presentation to PGRP-LE, similar to that observed in mammalian macrophages .
The PGRP domains of both PGRP-LC and PGRP-LE directly bind PGN in vitro, and ligand binding causes receptor oligomerization. PGRP-LC encodes three distinct receptors via alternative splicing: PGRP-LCa, PGRP-LCx and PGRP-LCy . Both PGRP-LCx and -LCa are involved in the recognition of TCT [7,14]. In particular, TCT binds in a deep PGN-binding cleft, typical of almost all PGRPs, in PGRP-LCx. However, the PGN-binding cleft in PGRP-LCa is occluded by short peptide insertions unique to PRGP-LCa . Instead, PGRP-LCa binds the complex of TCT–PGRP-LCx by interacting with a docking site created by the carbohydrate residues of TCT in combination with residues of PGRP-LCx that form the periphery of the deep PGN-binding cleft [15–17]. PGRP-LE also binds TCT and forms ligand-induced multimers via an almost identical mechanism . One notable difference between PGRP-LCa/x and PGRP-LE is that TCT induces an obligate heterodimer of LCx and LCa, because LCa cannot bind TCT, due to its occluded binding cleft, while TCT induces ‘infinite’ homo-multimers of PGRP-LE via a head-to-tail type Interaction. In both cases, it is likely that the TCT-induced multimers are critical for subsequent activation of signal transduction and the induction of AMPs.
PGRP-LCx and PGRP-LE both preferentially bind DAP-type PGN [19,20]. The response and binding characteristics of PGRP-LE are specific to DAP-type PGN, while PGPR-LCx exhibits some ability to weakly bind (and respond to) lysine-type PGN, in addition to its robust recognition of DAP-type PGN [17,21]. Preferential recognition of DAP-type PGN by PGRP-LC and PGRP-LE is produced through a similar molecular mechanism. Both PGRP-LCx and PGRP-LE include an arginine residue, which is found in the base of the PGN docking cleft of these receptors, that makes direct ionic contacts with the terminal (Cϵ) carboxy group of DAP [16,18]. Note that this carboxy group is the sole chemical difference distinguishing DAP from lysine. All DAP-type PGN-binding PGRPs, PGRP-LCx, PGRP-LE, PGRP-LB and human PGRP-S, include this conserved arginine residue, while lysine-specific PGRP-SA encodes a threonine in this position. Changing Arg254 in PGRP-LE to threonine dramatically reduces its affinity for TCT . In addition, Mariuzza and colleagues implicated two other conserved residues, Gly393 and Trp394 of PGRP-LCx, in the specific recognition of DAP-type PGN . Switching these residues to asparagine and phenylalanine, as found in PGRP-SA, allows for binding to a lysine-containing muropeptide . In the crystal structure, Trp394 is in close proximity to the DAP residue of TCT, forming part of the DAP-binding pocket. These interactions along with the key arginine salt bridge are critical for the preferential binding of DAP-type PGNs by this group of PGRPs.
On the other hand, PGRP-SA preferentially binds and responds to lysine-type PGNs [17,19,22]. Fruitflies lacking PGRP-SA fail to induce the AMP gene Drosomycin, a target of the Toll pathway, following infection with live Micrococcus luteus or injection of M. luteus (lysine-type) PGN [8,23]. DAP-type PGN more weakly stimulates the Toll pathway, but this also requires PGRP-SA and is consistent with the weak interaction between DAP-type PGN and PGRP-SA . In addition to preferential binding of lysine-type PGN, PGRP-SA may discriminate between DAP- and lysine-type PGN through a unique carboxypeptidase activity. Chang et al.  found that in vitro PGRP-SA specifically cleaves DAP-containing muropeptides (TCT), but not similar lysine-type muropeptides, hydrolysing the peptide bond between DAP and D-alanine . They speculate that this DAP-specific carboxypeptidase activity may selectively prevent activation of Toll signalling by DAP-type PGN.
Not all lysine-type PGN requires PGRP-SA recognition to stimulate the Toll pathway. Indeed, M. luteus appears to be unusual in its strict reliance on PGRP-SA. Many other Gram-positive, lysine-type PGN-producing bacteria are recognized through two partly redundant receptors: PGRP-SA and PGRP-SD. Only double PGRP-SA, PGRP-SD mutants fail to respond to Staphylococcus saprophyticus, Staphylococcus aureus, Streptococcus pyogenes and Enterococcus faecalis. It is not yet clear why M. luteus PGN, but not the others, is recognized only by PGRP-SA. The PGN structure of M. luteus is notably different from the others, including an unusual cross-bridging peptide sequence (which is identical with the stem-peptide sequence) and a glycine modification on the second position of the stem-peptide [1,24]. Perhaps PGRP-SA, but not PGRP-SD, is capable of binding this unusual PGN structure. Further work is required to address this question.
PGN recognition in the Toll pathway is further complicated by the role of GNBP1 (Gram-negative binding protein 1). GNBP1 was first identified based on its ability to bind Gram-negative bacteria, thus the moniker GNBP1, but, in fact, it is not involved in Gram-negative recognition in Drosophila [25–27]. Genetic studies have demonstrated that GNBP1 is required for the PGRP-SA-mediated recognition of Gram-positive bacteria and lysine-type PGN . Furthermore, Ligoxygakis and colleagues have demonstrated that polymeric lysine-type PGN must be processed into smaller fragments in order to activate Toll signalling through PGRP-SA [28,29]. They found that a muropeptide dimer (two disaccharide tetrapeptides cross-linked by a pentaglycine bridge) is the minimal active unit for Toll pathway activation and that GNBP1 is required to process polymeric PGN into these small, active muropeptides [28,29]. GNBP1 appears to function both by directly processing PGN and by interacting with PGRP-SA in a PGN-enhanced manner [29,30]. However, the enzymatic activity of GNBP1 appears to be limited to M. luteus PGN, and other lysozymes are likely to be involved in processing other PGNs, such as S. aureus PGN [29,30]. In addition, one of the catalytic PGRPs, PGRP-SC1a, is also implicated in the activation of the Toll pathway following S. aureus infection . Perhaps, PGRP-SC1a is also involved in processing PGN.
The receptors involved in detecting lysine-type PGN (PGRP-SA, PGRP-SD and GNBP1) function in the serum upstream of the cell-surface receptor Toll. In Drosophila, Toll functions as a cytokine receptor and its ligand is the protein Spätzle. Spätzle is made as a proprotein that is present in the haemolymph (the insect blood). Immune activation leads to proteolytic activation of Spätzle. PGN recognition by PGRP-SA, -SD and GNBP1 leads to the activation of a serine protease cascade that culminates in the cleavage of Spätzle. This pathway and the mechanisms involved in the PGRP-mediated protease cascade activation are not firmly established. However, it is clear that this pathway involves multiple CLIP-domain proteases and that the protease SPE (Spätzle processing enzyme) is responsible for cleaving Spätzle . Another CLIP-domain protease, Spirit, is likely to be involved in activating SPE. SPE and Spirit are also involved in Spätzle cleavage following fungal infection (which does not involve PGRP-mediated recognition). A third CLIP-domain serine protease known as Grass is uniquely required in the lysine-type PGN/PGRP pathway; however, the molecular link between this protease and the PGN–receptor complex is undefined . Once Spätzle is cleaved, it binds Toll and stimulates an NF-κB (nuclear factor κB) signalling pathway very similar to the MyD88 (myeloid differentiation factor 88)-dependent pathway used by most mammalian TLRs (Toll-like receptors) .
On the other hand, the receptors associated with the IMD pathway are associated with immune responsive cells and directly activate intracellular signal transduction. Except for the most receptor-proximal events, these intracellular signalling pathways will not be reviewed in detail here, but were described in a recent review . PGRP-LC and PGRP-LE interact with components of the intracellular IMD signal transduction cascade. In fact, a conserved motif, common to N-terminal signalling domains of both PGRP-LC and PGRP-LE, has been found to be essential for AMP gene induction downstream of these receptors . This conserved motif is weakly homologous with the RHIM [RIP (receptor-interacting protein) homotypic interaction motif] domain described in mammalian RIP1 and TRIF [TIR (Toll/interleukin-1 receptor) domain-containing adaptor protein inducing interferon β], which is critical for the interaction between these two proteins and signal transduction in the TRIF-dependent pathway downstream of TLR3 [35,36]. The mechanism by which the RHIM-like domain of PGRP-LC and -LE mediates signal transduction in the IMD pathway requires further investigation.
Our current understanding of the molecular mechanisms of PGN recognition in mammals is much less clear. Although early reports suggest that TLR2 might be a PGN receptor , more recent reports have argued that this PGN-mediated TLR2 activation was due to contaminants in the PGN preparations . However, this issue remains controversial with others still arguing that PGN, at high doses, can trigger TLR2 . It is widely accepted that several members of the NLR [NOD (nucleotide-binding oligomerization domain)-like receptor] family, including NOD1, NOD2 and NALP2/3, are triggered by small PGN-derived fragments . For example, MDP is an agonist of NOD2 and NALP2/3, and the PGN-derived dipeptide iE-DAP (D-glutamyl-meso-diaminopimelic acid) triggers NOD1. NOD1 and NOD2 stimulation activates NF-κB signalling pathways, while the NALPs are components of the inflammasome leading to caspase 1 activation and interleukin-1β processing and release. As with PGRP-LE, these NLRs function as intracellular receptors for small fragments of PGN. However, it is not clear if or how the NLRs recognize PGN. No direct binding of PGN fragments to these receptors has been reported and it has been suggested that co-receptors, which directly bind PGN, may be involved .
In contrast with insect PGRPs, which serve as PGN receptors for innate immune signalling pathways, mammalian PGRPs are directly bactericidal . Mammalian PGRPs are selectively expressed in tissues exposed to the environment, including the oral cavity, intestinal tract and skin. Human PRGPs are bactericidal against pathogenic and non-pathogenic Gram-positive bacteria, but not normal flora bacteria. Indeed, mammalian PGRPs are more potent, on a molar basis, than most antimicrobial peptides . As with certain glycopeptide antibiotics (e.g. vancomycin), PGRPs kill bacteria by directly interacting with their cell wall PGN, thereby interfering with PGN maturation. The crystal structure of human PGRP-Iβ in complex with GlcNAc-MurNAc-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala indicates that mammalian PGRPs disrupt cell wall maturation not only by sterically encumbering access of biosynthetic enzymes to the nascent PGN chains, but also by locking PGN into a conformation that prevents formation of cross-links between peptide stems in the growing cell wall .
In summary, the Drosophila immune system recognizes bacterial pathogens through the detection of PGN. At least four members of the PGRP family of receptors are involved in PGN detection in fruitflies. These recognition receptors are non-catalytic members of the PGRP family. On the other hand, the amidase PGRPs are best characterized for the role in degrading PGN, thereby down-modulating the immune response [44,45]. Different types of PGN are recognized by different receptors. This differential recognition leads to the activation of different immune response pathways, the IMD and Toll pathways, which in turn have overlapping and distinct outputs [46,47]. Through crystallographic analyses, the mechanisms of preferential recognition of DAP-type PGN by PGRP-LC and LE is well characterized, while better characterization of the PGN binding activities of PGRP-SA and -SD, and a more detailed analysis of its carboxypeptidase activity, are needed to understand the preferential activation of the Toll pathway by lysine-type PGN. In addition, many unanswered questions remain on the molecular mechanisms that link these receptors to their downstream signalling cascades.
Pattern-Recognition Receptors in Human Disease: A Biochemical Society Focused Meeting held at Queens' College, University of Cambridge, Cambridge, U.K., 8–10 August 2007. Organized and Edited by C. Bryant (Cambridge, U.K.), K. Fitzgerald (University of Massachusetts Medical School, U.S.A.), N. Gay (Cambridge, U.K.), P. Morley (GlaxoSmithKline, U.K.) and L. O'Neill (Trinity College Dublin, Ireland).
Abbreviations: AMP, antimicrobial peptide; DAP, diaminopimelic acid; GNBP, Gram-negative binding protein; IMD, immune deficiency; MDP, muramyl-dipeptide; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor κB; NOD, nucleotide-binding oligomerization domain; NLR, NOD-like receptor; PGN, peptidoglycan; PGRP, PGN recognition protein; RIP, receptor-interacting protein; RHIM, RIP homotypic interaction motif; SPE, Spätzle processing enzyme; TCT, tracheal cytotoxin; TIR, Toll/interleukin-1 receptor; TLR, Toll-like receptor; TRIF, TIR domain-containing adaptor protein inducing interferon β
- © The Authors Journal compilation © 2007 Biochemical Society