InteractiveFly: GeneBrief

Peptidoglycan recognition protein LC: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila antimicrobial response is one of the best characterized systems of pattern recognition receptor-mediated defense in metazoans. Drosophila senses Gram-negative bacteria via two peptidoglycan recognition proteins (PGRPs), membrane-bound PGRP-LC and secreted/cytosolic PGRP-LE, which relay diaminopimelic acid (DAP)-type peptidoglycan sensing to the Imd signaling pathway. In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their peptidoglycan binding specificities. This study used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of PGRP-LC, and the tissue-specific roles were assessed of PGRP-LC isoforms and PGRP-LE in the antibacterial response. In vivo studies demonstrate the key role of PGRPLCx in sensing DAP-type peptidoglycan-containing Gram-negative bacteria or Gram-positive bacilli during systemic infection. The contribution of PGRP-LCa/x heterodimers to the systemic immune response to Gram-negative bacteria was highlighted through sensing of tracheal cytotoxin (TCT), whereas PGRP-LCy may have a minor role in antagonizing the immune response. The results reveal that both PGRP-LC and PGRP-LE contribute to the intestinal immune response, with a predominant role of cytosolic PGRP-LE in the midgut, the central section of endodermal origin where PGRP-LE is enriched. The in vivo model also definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a loss-of-tolerance model. In conclusion, this study delineates how a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila (Neyen, 2012).

In animals, the innate immune system detects bacterial infection through the use of germline-encoded pattern recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs), such as LPS, peptidoglycan, or flagellin. After the identification of PRRs and their respective ligands, a challenge in the field is to understand how each of these various PRRs contributes to an effective and adapted immune response. The study of innate immune recognition is complicated by the existence of multiple PRRs with various expression patterns, variation in PAMP exposure, and modifications through the action of host and bacterial enzymes during the course of infection. In addition, PAMP signals intersect with a less well understood but equally complex network of endogenous danger signals, which allow the immune system to discriminate between pathogenic and non-pathogenic microorganisms. A better understanding of the mode of action of PRRs ideally requires an in vivo approach in whole organisms using natural routes of infection. The Drosophila antimicrobial response is one of the best characterized systems of PRR-mediated defense in metazoans and provides a good model to understand both the logic of pattern recognition and how PRRs shape the ensuing immune response. This study used Phi31-mediated recombineering to generate fly lines expressing specific isoforms of peptidoglycan recognition protein (PGRP)-LC, a Drosophila PRR involved in sensing Gram negative bacteria (Neyen, 2012).

Pattern recognition upstream of the two Drosophila innate immune response branches, the Toll and Imd pathways, relies to a large extent on peptidoglycan sensing by PGRPs. Peptidoglycan, a cell wall component found in almost all bacteria, is a polymer of alternating N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), cross-linked by short peptide bridges whose amino acid composition and organization differs among bacteria. As evidenced by Gram staining, peptidoglycan (PGN) forms an abundant external layer in Gram-positive bacteria but is less abundant in Gram-negative bacteria where it is hidden under an external layer of LPS. The structure of PGN from Bacillus and Gram-negative bacteria differs from that of most Gram-positive PGN in the third amino acid position of the peptide bridge. Gram-negative and Bacillus-type PGNs are cross-linked by a peptide containing a meso-diaminopimelic residue, whereas in other Gram-positive bacterial PGNs a lysine is found in this position. In addition, diaminopimelic acid (DAP)-type PGN from Gram-negative bacteria but not DAP-type bacilli contains anhydro-MurNAc residues at the end of each PGN strand, which are distinctive footprints of bacterial PGN synthetic enzymes. Monomers of GlcNAc-1,6-anhydro-MurNAc-L-Ala-γ-D-Glu-meso- DAP-D-Ala, also called tracheal cytotoxin (TCT), represent ~5% of GlcNAc-MurNAc residues and were previously shown to be the minimal PGN motif to elicit Imd responses in flies. As anhydro-muropeptides are released during bacterial cell wall synthesis, TCT has been put forward as a specific indicator of potentially dangerous Gram-negative bacterial proliferation (Neyen, 2012).

Use of highly purified products has demonstrated that in contrast to vertebrates, sensing of Gram-negative bacteria in Drosophila is not based on recognition of LPS. Rather, the ability of Drosophila to discriminate between Gram-positive and Gram-negative bacteria relies on the recognition of specific forms of PGN by PGRPs. The Drosophila genome carries a total number of 13 PGRP genes, which give rise to 19 known different receptors. The family comprises both enzymatically active, generally secreted, amidase PGRPs that cleave PGN into non-immunogenic fragments and catalytically inactive receptors, generally membrane-bound, which mediate ligand-dependent downstream signaling (Werner, 2000). All family members contain at least one PGRP domain, which is structurally related to bacterial T7 lysozymes and recognizes different types of PGN. Whereas PGRP-SA upstream of Toll recognizes mainly lysine-containing PGN from Gram-positive bacteria, Imd-activating PGRP-LC and PGRP-LE exclusively sense DAP-type PGN from Gram-negative bacteria and Gram-positive bacilli (Leulier, 2003). In the case of PGRP-LC, differential splicing of PGRP domain-encoding exons to a common intracellular domain-encoding exon generates three receptor isoforms, which differ in their PGN binding specificities but share identical signaling capacities (Kaneko, 2004; Werner, 2003). PGRP-LF, a highly similar but signaling-deficient receptor encoded by the locus adjacent to PGRP-LC, contains two functional PGRP domains but lacks the intracellular signaling domain and acts as a negative regulator of Imd activation. Crystal structures of ligand-binding domains of PGRP-LC isoforms in the presence of monomeric PGN have defined the molecular basis for ligand binding. Only PGRP-LCx contains the characteristic L-shaped PGN binding groove described for mammalian PGRP-Ia (Guan, 2004) and Drosophila PGRP-LB (Kim, 2003) and can accommodate polymeric and monomeric PGN. Protruding residues in the ligand binding pocket of PGRPLCa prevent direct binding of TCT (Chang, 2005; Chang, 2006), but PGRP-LCa dimerizes with PGRP-LCx-TCT complexes via its PGN binding groove (Chang, 2006; Mellroth, 2005). Notably, PGRP domain affinity studies have determined equivalent binding constants for PGRP-LCa and PGRPLF to PGRP-LCx-TCT complexes (Basbous, 2011). Because activation of the Imd pathway relies on ligand-induced receptor homo- or hetero-multimerization, this implies that the stoichiometry of signaling-efficient PGRP-LC isoforms to the signaling-deficient PGRP-LF determines the strength of pathway activation (Neyen, 2012).

Studies in cell culture using RNA interference (RNAi) specific for each PGRP-LC isoform have shown that PGRP-LCx is required for recognition of polymeric PGN, whereas both PGRPLCa and PGRP-LCx are mandatory for detection of monomeric PGN (Kaneko, 2004; Werner, 2003). It has been proposed that signaling is achieved by association of at least two PGRP-LCx molecules in close proximity through binding of polymeric PGN (Choe, 2005). Such an interaction cannot occur with monomeric PGN, and in this case PGRP-LCa is expected to act as an adapter (Mellroth, 2005). This model is supported by the crystallization of TCT in complex with both PGRP-LCa and PGRP-LCx (Neyen, 2012).

Although loss-of-function mutants established the fundamental role of PGRP-LC in survival to Gram-negative infection (Choe, 2002; Gottar, 2002; Ramet, 2002), the residual antimicrobial peptide response in flies lacking PGRP-LC compared with Imd-deficient flies suggested a second receptor upstream of Imd. PGRP-LE encodes a PGRP with affinity to DAP-type PGN and is expressed both extracellularly and intracellularly. A secreted fragment of PGRP-LE corresponding to the PGRP domain alone functions extracellularly to enhance PGRP-LC- mediated PGN recognition on the cell surface, a role evocative of that of mammalian CD14 in binding of LPS to TLR4. The full-length form of PGRP-LE is cytoplasmic and acts as an intracellular receptor for monomeric PGN, effectively bypassing the requirement for PGRP-LC (Kaneko, 2006). Thus, both PGRP-LC and PGRP-LE account for sensing of Gram-negative bacteria upstream of the Imd pathway. Finally, detection of DAP-type PGN in Drosophila is modulated by amidase PGRPs, which enzymatically degrade PGN and reduce the amount of available immunostimulatory compounds. Among these, PGRP-LB has been best characterized as a negative regulator of the Imd pathway (Zaidman-Remy, 2006). Despite a wealth of studies, several questions remain to be addressed, including the respective contribution of each PGRP-LC isoform and PGRP-LE in response to bacteria as well as the differential requirement of these PRRs in specific tissues, especially in barrier epithelia such as the gut that are constantly exposed to bacterial stimuli. Overexpression studies using full-length and ectodomain-truncated receptors lead to ligand-independent activation of the immune response, probably due to increased receptor proximity in the membrane (Choe, 2005). It is therefore crucial to use an in vivo model with wild-type receptor levels to interpret correctly the mechanism of ligand-specific Imd activation downstream of various PGRP-LC isoforms. This study therefore used a genomic complementation approach to supplement PGRP-LC-deficient mutants with isoform-specific PGRP-LC loci elsewhere in the genome (Neyen, 2012).

This approach allowed generation of wild-type levels of defined PGRP-LC isoforms in vivo and to assess the tissue-specific roles of each isoform, alone or in various combinations. The results confirm previously described roles of PGRP-LCx and PGRP-LCa/x dimers in polymeric and monomeric PGN sensing, respectively, and uncover a new role for PGRP-LE in the activation of the Imd pathway in the gut. In addition, the in vivo model definitively establishes TCT as the long-distance elicitor of systemic immune responses to intestinal bacteria observed in a case of rupture of tolerance induced by knockdown of amidase PGRP-LB (Neyen, 2012).

The initial aim of this study was to define the role of each PGRP-LC isoform in vivo. Using Phi31-mediated recombineering, loci of full and isoform-specific PGRP-LC constructs were successfully inserted into the fly genome, and they were proved capable of complementing PGRP-LC null mutations. The in vivo approach confirms and extends previous in vitro and RNAi experiments in proving that PGRP-LCx is indeed necessary and sufficient to respond to challenge with live or dead Gram-negative bacteria and to Gram-positive, DAP-type bacilli. Moreover, PGRP-LCx alone induces the in vivo immune response to polymeric PGN, whereas combined presence of PGRP-LCx and PGRP-LCa is necessary to sense the anhydro-monomer TCT. The differential requirement of PGRP-LC isoforms in response to Gram-positive DAP-type (PGRP-LCx alone) and Gram-negative bacteria (both PGRP-LCx and PGRP-LCa/x) indicates that flies are able to discriminate between the two types of DAP-type PGN-containing bacteria and to mount appropriate responses. Notably, injection of TCT in contrast to polymeric PGN leads to an increase in amplitude and duration of Imd pathway activation. Thus, TCT detection by PGRP-LCa/x allows flies to mount a strong response to Gram-negative bacteria despite the fact that DAP-type PGN is not exposed (masked by the LPS layer) and is less abundant compared with DAP-type PGN-containing Gram-positive bacteria (Neyen, 2012).

Consistent with previous reports that showed no effect of PGRPLCy RNAi on PGN sensing in cells, PGRP-LCy on its own did not show any induction of the Imd pathway. However, bacterial infection or injection of immunostimulatory compounds repeatedly produced a stronger response in flies carrying PGRPLCa/ x isoforms than in flies carrying the whole PGRP-LC locus. Although subtle differences in isoform expression from the intact, full locus compared with the engineered isoform loci cannot be excluded, this suggests that the full locus carries an additional regulatory element lacking in heterozygous PGRP-LCa/x flies. It is tempting to speculate that PGRP-LCy, present in the full locus but absent from PGRP-LCa/x flies, might help to regulate response levels. PGRP-LCy is structurally unlikely to bind PGN but, unlike PGRP-LF, retains a signaling-competent cytoplasmic tail. If any regulatory activity was associated with the PGRP-LCy isoform, it would therefore have to act extracellularly, possibly by competing with other isoforms for cell surface localization and thereby diluting receptor availability. Thus, the only function that can be attributed to PGRP-LCy from this study is a regulatory role in adjusting the amplitude of Imd pathway activation. The importance of wild-type receptor levels in any study of isoform function is crucial because overexpression of receptors is sufficient on its own to stimulate the Imd pathway. The PGRP-LC complemented system mimics wild-type receptor expression dynamics, and no elevated background levels of Imd activation was detected in complemented PGRP-LC mutant flie. However, alterations in the genomic ratio of PGRP-LC to PGRP-LF, achieved by combining [LC] or [LC,LF] vector-carrying lines with wild-type or different PGRP-LC-deficient backgrounds, showed a significant correlation between Dpt levels and PGRP-LC/LF ratios in infected flies, consistent with an inhibitory role of PGRP-LF. This indicates that the stoichiometry of activating and regulating receptors matters, as foreshadowed by affinity studies between signaling-competent PGRP-LCx-TCT-PGRP-LCa and signaling-deficient PGRPLCx-TCT-PGRP-LF complexes (Neyen, 2012).

Several overexpression studies in S2 cells already localized PGRP-LC to the plasma membrane. This study extend this finding to wild-type receptor expression levels in an immunocompetent tissue and provides evidence that PGRP-LC localizes to the apical and lateral plasma membrane in fat body cells, revealing a previously undescribed polarity in this immune-responsive tissue. Similar to Takehana (2004), who described no significant difference between Diptericin expression in PGRP-LC versus PGRP-LE;; PGRP-LC mutants after stimulation with B. subtilis and Escherichia coli, no additional decrease was seen in survival rates to Erwinia carotovora carotovora 15 when comparing single PGRP-LC and double PGRP-LE;;LC mutants, and no significant underlying reduction in Diptericin levels. This underlines the major role of PGRP-LC to survey a defined compartment -- the insect hemolymph -- and to preferentially activate immune responses in the fat body. In general agreement with Kaneko (2006), this study confirmed a role of PGRP-LE in the systemic immune response to TCT, albeit depending on the route of administration. On one hand, we note a predominant role of PGRP-LCa/x over PGRP-LE in sensing injected TCT in the hemolymph. In this context, the contribution of PGRP-LE was discernible in the presence of any PGRP-LC isoform but was less marked in the absence of the full locus, consistent with the concept that hemolymph PGRP-LE cannot signal directly but depends on membrane-bound PGRP-LC to relay information (Kaneko, 2006). However, even though secreted PGRP-LE might contribute to Imd activation by delivering hemolymph TCT/ PGN to membrane-bound PGRP-LC, the effect of complete PGRP-LE loss on systemic immune activation after injection of TCT into the hemocele was not significant. This suggests that the cytosolic, autonomous PGRP-LE form does not contribute significantly to the activation of the Imd pathway by injected TCT and establishes PGRP-LC as the predominant receptor eliciting systemic responses in the hemocele (Neyen, 2012).

In contrast, when Imd activation in the fat body was triggered by oral ingestion of TCT in the PGRP-LB mutant background, a non-negligible contribution of PGRP-LE was observed to this systemic response in the absence of PGRP-LC. This indicates that when TCT reached the hemocele by active or passive transport from the intestine, the role of cytosolic PGRP-LE became more prominent. Although these is no explanation for this discrepancy, one might speculate that even though cytosolic PGRP-LE does not significantly contribute to TCT sensing when injected into the hemolymph, possibly because the fat body lacks transporters present in absorptive organs, this intracellular mode of recognition gains in importance when TCT transits through cells. Taken together, the subordinate role of secreted PGRP-LE compared with PGRP-LC might suggest that the main contribution of PGRP-LE is as an intracellular sensor, which will only spring into action when systemic levels of TCT have reached a critical threshold and permeated the cytosol (Neyen, 2012).

Determining the mechanisms by which barrier epithelia sense bacteria and differentiate between acceptable and non-acceptable intruders is a major issue in the field of innate immunity. Previous studies proposed PRR compartmentalization as an essential mechanism to discriminate between pathogenic versus beneficial bacterial colonization. Although this study observed a clear role of PGRP-LC sensing in the gut, consistent with previous studies, it is not possible to conclude whether this reflects direct sampling of the gut lumen by PGRP-LC. Unfortunately, the expression of the PGRPLC- GFP fusion construct was not strong enough to determine whether PGRP-LC is expressed at the apical or the basal side of enterocytes. Of note, recognition PGRPs involved in the sensing of Gram-negative bacteria show differential expression patterns along the gut, with enrichment of PGRP-LE in the endodermally derived midgut and a modest enrichment of PGRP-LC in ectodermally derived foregut and hindgut. Moreover, PGRPs in these sections are more or less accessible to gut contents. A relatively impermeable cuticle protects ectodermal epithelia in the foregut and hindgut, whereas the peritrophic matrix covering the PGRP-LE- rich section of the midgut is permeable to allow passage of digested nutrients. It is therefore more likely for bacterial compounds to reach midgut epithelia, and a reduction in surface receptors capable of mounting potentially detrimental immune responses to commensals in this compartment would make sense. Cytosolic receptors expressed in this compartment would be able specifically to detect absorbed or diffusible bacterial compounds such as TCT, which may be a hallmark of proliferation and/or harmful bacteria. Consistent with this, a major contribution of PGRP-LE (most probably of the cytosolic form as PGRP-LE signaling did not depend on PGRP-LC) and less of PGRP-LC when the midgut-specific response to Gram-negative bacteria was assessed. More strikingly, the midgut response to ingested TCT relied mostly on PGRP-LE, supporting a role of this receptor in danger detection in the gut. Thus, this study uncovers a key role of PGRP-LE in the Drosophila midgut and suggests that intracellular sensing of TCT is used in Drosophila as a mechanism to recognize infectious bacteria (Neyen, 2012).

Previously a model was put forward whereby long-range activation of the systemic immune response in Drosophila is mediated by the translocation of small PGN fragments from the gut lumen or other barrier epithelia to the hemolymph. This view was supported by the observation that ingestion of monomeric PGN can stimulate a strong systemic immune response in PGRP-LB knockdown flies with reduced amidase activity and that deposition of PGN or TCT on the genitalia is sufficient to induce a systemic immune response. Moreover, because TCT consistently elicited stronger responses than PGN, these models proposed an involvement of active or passive transport of the elicitor to the hemocele. On the basis of the current results, the mechanism of TCT delivery to the hemocele is still uncertain. However, the unique and well-characterized interaction of TCT-PGRP-LCa-PGRPLCx (Chang, 2006) and the primordial role of PGRP-LCa/x heterodimers in mediating TCT-specific systemic activation of the Imd pathway demonstrates that TCT is indeed a crucial element in the long-range activation of the immune response (Neyen, 2012).

In conclusion, this study shows that a combination of extracellular sensing by PGRP-LC isoforms and intracellular sensing through PGRP-LE provides sophisticated mechanisms to detect and differentiate between infections by different DAP-type bacteria in Drosophila. It is probable that the absence of LPS sensing in Drosophila has imposed some constraints on the system and that sensing of TCT through PGRP-LCa/x and PGRP-LE evolved as a surrogate way to distinguish Gram-negative bacteria from Gram-positive DAP-type PGN-containing bacteria. Because TCT is released during bacterial division, intracellular sensing through PGRP-LE provides an adequate mechanism of detection in the gut, reminiscent of the intracellular sensing of Gram-negative muropeptides by intracellular NOD1 in epithelia. To date, the existence of a mode of recognition of lysine-type bacteria in the midgut remains unexplored. A simple explanation could be that lysine-type bacteria do not represent a threat for flies as they rarely infect via the oral route and are therefore not detected. Indeed, DAP-type PGN-containing bacteria of either Gram-negative type (Serratia, Pseudomonas) or bacillus-type (Bacillus thuringiensis) are the only characterized naturally occurring insect pathogens to date (Neyen, 2012).

The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity

It is now appreciated that the brain is immunologically active. Highly conserved innate immune signaling responds to pathogen invasion and injury and promotes structural refinement of neural circuitry. However, it remains generally unknown whether innate immune signaling has a function during the day-to-day regulation of neural function in the absence of pathogens and irrespective of cellular damage or developmental change. This study shows that an innate immune receptor, a member of the peptidoglycan pattern recognition receptor family (PGRP-LC), is required for the induction and sustained expression of homeostatic synaptic plasticity. This receptor functions presynaptically, controlling the homeostatic modulation of the readily releasable pool of synaptic vesicles following inhibition of postsynaptic glutamate receptor function. Thus, PGRP-LC is a candidate receptor for retrograde, trans-synaptic signaling, a novel activity for innate immune signaling and the first known function of a PGRP-type receptor in the nervous system of any organism (Harris, 2015).

The homeostatic modulation of presynaptic neurotransmitter release has been observed at mammalian central synapses and at neuromuscular synapses in species ranging from Drosophila to mouse and human. The homeostatic enhancement of presynaptic release following inhibition of postsynaptic glutamate receptors is achieved by an increase in presynaptic calcium influx through presynaptic CaV2.1 calcium channels and the simultaneous expansion of the readily releasable pool (RRP) of synaptic vesicles. To date, the retrograde, trans-synaptic signaling system that initiates these presynaptic changes following disruption of postsynaptic glutamate receptors remains largely unknown (Harris, 2015).

A large-scale forward genetic screen for homeostatic plasticity genes, using synaptic electrophysiology at the neuromuscular junction (NMJ) as the primary assay, identified mutations in the PGRP-LC locus that block presynaptic homeostasis. In Drosophila, PGRP-LC is the primary receptor that initiates an innate immune response through the immune deficiency (IMD) pathway. In mammals, there are four PGRPs including a 'long' isoform (PGRP-L or PGlyRP2) that appears to have both secreted and membrane-associated activity. In mice, the PGlyRP2 protein has two functions in the innate immune response: a well-documented extracellular enzymatic (amidase) activity and a pro-inflammatory signaling function that is independent of its extracellular enzymatic activity. As yet, there are no known functions for PGRPs in the nervous system of any organism (Harris, 2015).

Innate immune signaling has been found to participate in neural development and disease including a role for the C1q component of the complement cascade, Toll-like receptor signaling, and tumor necrosis factor signaling. In these examples, however, the innate immune response is induced within microglia or astrocytes. Far less clear is the role of innate immune signaling within neurons, either centrally or peripherally, although there are clear examples of downstream signaling components such as Rel and NFκB having important functions during learning related neural plasticity. This study places the innate immune receptor, PGRP-LC at the presynaptic terminal of Drosophila motoneurons and demonstrate that this receptor is essential for robust presynaptic homeostatic plasticity. It is speculated, based on the current data, that PGRP-LC could function as a receptor for the long-sought retrograde signal that mediates homeostatic signaling from muscle to nerve (Harris, 2015).

A distinguishing feature of presynaptic homeostatic plasticity is that it can be both rapidly induced and sustained for prolonged periods. It is possible that innate immune signaling activity is adjusted to fit the requirements of presynaptic homeostasis in motoneurons. This would be consistent with the established diversity of innate immune signaling functions during development such as dorso-ventral patterning. Alternatively, the induction of innate immune signaling might serve a unique function during presynaptic homeostasis. Many homeostatic signaling systems incorporate feed-forward signaling elements. By analogy, PGRP-LCx could function as a feed-forward signaling receptor that acts more like a 'switch' to enable presynaptic homeostasis in the nerve terminal. The accuracy of the homeostatic response would be determined by other signaling elements. In favor of this idea, the induction of innate immune signaling is rapid, occurring in seconds to minutes, and can be maintained for the duration of an inducing stimulus, consistent with recent evidence that presynaptic homeostasis is rapidly and continually induced at synapses in the presence of a persistent postsynaptic perturbation. Finally, it remains formally possible that PGRP-dependent signaling is a permissive signal that allows expression presynaptic homeostasis (Harris, 2015).

Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviours that lower the impact of the infection. The molecular mechanisms by which microbes impact host behaviour are not well understood. This study demonstrated that bacterial infection of Drosophila females reduces oviposition and that bacterial cell wall peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. Peptidoglycan regulates egg laying rate by activating PGRP-LC -> NF-κB (Relish) signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioural response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioural immunity (Kurz, 2017).

In addition to activate direct antimicrobial strategies, eukaryotes have developed behavioral mechanisms that facilitate the avoidance of pathogens or lower the impact of the infection. These phenotypes grouped under the term 'behavioral immunity' or 'sickness behavior' refer to a suite of neuronal mechanisms that allow organisms to detect the potential presence of disease-causing agents and to engage in behaviors which prevent contact with the invaders or reduce the consequences of the infection. Although such microbe-induced behavioral changes have been reported in Lepidoptera and Orthoptera, deciphering the molecular mechanisms involved is experimentally challenging in these insects. Indeed, such an analysis requires a model organism with genetic tools allowing the manipulation of actors and regulators of both the immune and neuronal systems. Recent reports, mainly in Drosophila, start to unravel some aspects of these peculiar host-microbe interactions. Stensmyr et al. demonstrated that Drosophila avoid food contaminated by pathogenic bacteria by using an olfactory pathway exquisitely tuned to a single microbial odor, Geosmin (Stensmyr, 2012).

Produced by harmful microorganisms, Geosmin is detected by specific Drosophila olfactory sensory neurons which then transfer the message to higher brain centers. Activation of this olfactory circuit ultimately induces an avoidance response, and suppresses egg-laying and feeding behaviors, thereby reducing the infection risk of both the adult flies and their offspring. Drosophila not only modify their behavior to avoid contamination by microbes or parasites, but also once they have been contaminated in order to reduce the impact of infection. For instance, direct exposure to bacteria impacts sleep patterns and induces hygienic grooming. In addition, Drosophila plastically increases the production of recombinant offspring in response to parasite infection. Although certainly involving a neuro-immunological integration, these microbe-induced behavioral changes are rarely understood at the molecular level, namely with no information on the nature of the elicitor and on the cellular and molecular machineries that link bacteria detection to behavioral changes. Moreover, canonical immune signaling pathways were never reported as being involved in those processes (Kurz, 2017).

The data demonstrate that bacteria derived cell wall peptidoglycan (PGN) entry into the fly body cavity has, at least, two physiological consequences. In addition to activate innate immune response in fat body cells, it also blocks mature egg delivery in oviduct and hence reduces egg laying of infected females. It was further demonstrated that this bacterially induced behavioral change is due to an NF-κB pathway-dependent modulation in octopaminergic neurons. Evidence is presented that both responses, that are potentially detrimental if not down-regulated, are fine-tuned by distinct and specific PGN degrading enzymes. It is proposed that by regulating the level of internal PGN, flies adapt their egg-laying behavior to environmental conditions. In standard environmental conditions, PGRP-LB ensures that low level of PGN does not affect egg laying. However, whenever PGN concentration reaches a certain threshold, which either reflects an infection status or the presence of a highly contaminated food supply, NF-κB pathway activation in neurons is blocking egg release. As PGN of ingested bacteria is capable of reaching the internal fluid and triggering dedicated signaling cascades, one could imagine that such a mechanism prevents flies from laing their eggs in highly contaminated food, in which their development and that of the hatching larvae could be impaired by microbes. In this context, PGRP-LB mediated PGN scavenging is crucial since a non-regulated behavioral immune response would lead to a severe drop in the amount of progeny which may not be in keeping with the real threat. Another possibility could be that a reduced egg production will favor immune effector production. Indeed, it is often considered that the energy cost of an acute innate immune response needs can be balanced by a decreased offspring production. Blocking the energy-consuming egg production in infected flies could be a way for them to mobilize resources required for full activation of innate immune defences. A similar depression of oviposition has recently been documented in females flies exposed to parasitoid wasps who lay their eggs in Drosophila larvae. However, while visual perception of wasps by female flies induces a long-term decline in oviposition associated with an early stage-specific oocyte apoptosis, PGN effects are transient and rather lead to a late stage oocyte accumulation suggesting that although the final outcome is the same, the mechanisms differ (Kurz, 2017).

The data from this study indicate that PGN sensing acts on egg-laying behavior via neuronal modulation. NF-κB pathway signaling in octopaminergic neurons was identified as the actor of this PGN-dependent oviposition reduction. It would be informative to test whether bacterial infection is also affecting other octopamine-mediated behaviors such as reward in olfactory or visual learning, male-male courtship, male aggressive behavior. This would require to further characterize the nature of the octopamine neurons whose activation is modulated by infection and to consider that the phenotypes defined as being part of the sickness behaviours might be orchestrated directly by the immune system following the perception of microbes. Indeed, a PGRP-LB^PD reporter line not only labels cells in the reproductive tract but also in thoraco-abdominal ganglia and in the brain with projections to proboscis, wings and legs. Likewise, octopaminergic neurons have been shown to innervate numerous areas in the brain and in the thoraco-abdominal ganglion and to project to various reproductive structures such as ovaries, oviducts and uterus, further work will be needed to exactly pinpoint the identity of the affected octopaminergic neurons, their targets and their effect on fly behavior. In addition, the question remains as to how NF-κB activation can modulate octopaminergic neurons activity. Among the possibilities is the modulation of octopamine neuron excitability, the regulation of octopamine production or its secretion. Knowing the NF-κB protein itself is required for this behavioral response and that increasing the amount of available octopamine via overexpression of the TβH enzyme rescues the oviposition drop, it is expected that IMD pathway activation in neurons will have transcriptional consequences. However, other hypotheses might be considered since Dorsal, a member of the other Drosophila NF-κB signaling cascade Toll, has been shown to function post-transcriptionally together with IκB and IRAK at the post-synaptic membrane to specify glutamate receptor density. It should also be noticed that PGRP-LC has recently been shown to control presynaptic homeostatic plasticity in mouse (Harris, 2015). One of the future challenges will be to understand how NF-κB activation is reducing octopaminergic signals (Kurz, 2017).

This study shows that Drosophila uses an unique bacteria associated molecular pattern to activate different processes related to host defence, namely the production of antimicrobial peptides and the modulation of oviposition behavior. Interestingly, it appears that in order to fine-tune these responses, different isoforms of the same PGN scavenging enzyme, PGRP-LB, are required. While the secreted PGRP-LB^PC isoform certainly acts non cell-autonomously to dampen immune activation by circulating PGN, a putatively intracellular isoform PGRP-LB^PD controls the effect of PGN on oviposition. Even more remarkable, this response is not transmitted via PGRP-LC but rather by the intracytoplasmic PGRP-LE receptor. Previous work has shown that PGRP-LE is also regulating response to bacteria in some part of the gut. Thus, it will be important to understand how PGN is trafficking within and through cells, and how PGRP-LB^PD modulates PGRP-LE-dependent IMD pathway activation and whether it is also required to modulate other PGN/PGRP-LE-dependent responses (Kurz, 2017).

In essence, the results demonstrate that PGN, when ingested or introduced into the body cavity, not only activates antibacterial immune response but also influences neuronally controlled behaviors in flies. Importantly, the sickness behavior deciphered in this study does not appear to be a side effect of an energetically expensive immune response, but rather the result of a specific regulation. An orchestration of different processes required for the immune response was also exemplified by a recent report linking metabolism and immunity. Although not dissected to the molecular level, previous studies in mammals have suggested that similar interactions between PGN and neuronally controlled activities. For instance, PGN derived muropeptide MDP has been shown to display powerful somnogenic effect when injected into rabbit braint. It has also been shown that PGN produced by symbiotic microbiota may 'leak' into the bloodstream and reach organs distant to the gut, such as the bones. Finally, recent findings show that bacterial cell wall peptidoglycan traverses the murine placenta and reach the developing fetal brain where it triggers a TLR2-dependent fetal neuroproliferative response. A future challenge will be to test whether an NF-κB-dependent response to PGN is also taking place in mammalian neurons and directly influences the animal behavior (Kurz, 2017).

The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors

The innate immune system needs to distinguish between harmful and innocuous stimuli to adapt its activation to the level of threat. How Drosophila mounts differential immune responses to dead and live Gram-negative bacteria using the single peptidoglycan receptor PGRP-LC is unknown. This study describes rPGRP-LC, an alternative splice variant of PGRP-LC that selectively dampens immune response activation in response to dead bacteria. rPGRP-LC-deficient flies cannot resolve immune activation after Gram-negative infection and die prematurely. The alternative exon in the encoding gene, here called rPGRP-LC, encodes an adaptor module that targets rPGRP-LC to membrane microdomains and interacts with the negative regulator Pirk and the ubiquitin ligase DIAP2. rPGRP-LC-mediated resolution of an efficient immune response requires degradation of activating and regulatory receptors via endosomal ESCRT sorting. It is proposed that rPGRP-LC selectively responds to peptidoglycans from dead bacteria to tailor the immune response to the level of threat (Neyen, 2016).

PGRP-LC has a clear role as the major signaling receptor sensing Gram-negative bacteria in flies, but its contribution to the resolution phase once bacteria are killed and release polymeric PGN has remained elusive. This study has uncovered a regulatory isoform of LC (rLC) that adjusts the immune response to the level of threat. rLC specifically downregulates IMD pathway activation in response to polymeric PGN, a hallmark of efficient bacterial killing. The data are consistent with a model whereby the presence of rLC leads to efficient endocytosis of LC and termination of signaling via the ESCRT pathway. Trafficking-mediated shutdown of LC-dependent signaling ensures that LC receptors are switched off once the balance is tipped in favor of ligands signifying dead bacteria, allowing Drosophila to terminate a successful immune response. Failure to do so results in over-signaling, leading to the death of the host despite bacterial clearance. Consistent with this model, defects were found in endosome maturation and in the formation of MVBs enhance immune activation and prevent immune resolution. In addition to regulating LC signaling via the ESCRT machinery, rLC can also inhibit LC signaling by forming signaling-incompetent rLC-LC heterodimers or rLC-rLC homodimers (Neyen, 2016).

Recent evidence from vertebrates also implicates the ESCRT machinery in suppressing spurious NF-κB activation: the TNFR superfamily member lymphotoxin-β receptor, which activates a signaling cascade that is functionally similar to IMD signaling, is degraded in an ESCRT-dependent manner in zebrafish and human cells. Thus ESCRT-mediated clearance of receptors upstream of NF-κB seems well conserved throughout evolution (Neyen, 2016).

Internalization of receptor-ligand complexes raises the question of whether peptidoglycan is fully degraded in the endolysosomal compartment or fragmented and released into the cytosol for sensing by PGRP-LE, as is the case for peptidoglycan sensing by cytoplasmic NOD2 receptors in mammalian cells. Mechanistic coupling of LC-dependent peptidoglycan endocytosis and PGRP-LE-dependent cytosolic sensing of exported peptidoglycan fragments would help explain the partial cooperation between the two receptors (Neyen, 2016).

Molecularly, rLC is characterized by a cytosolic PHD domain predicted to bind to phosphoinositides. The PHD domain targets rLC were found to be distinct membrane domains but it cannot be excluded that this localization relies on additional protein-protein interactions. Furthermore, the PHD domain also mediates binding of rLC to the cytosolic regulator Pirk and the ubiquitin ligase DIAP2. The combined capability to control membrane localization and to recruit downstream signaling modulators is reminiscent of the 'sorting-signaling adaptor paradigm' that is emerging for mammalian PRRs. Sorting adaptors are cytosolic signaling components with phosphoinositide-binding domains that are selectively recruited to defined subcellular locations and thereby shape the signaling output of the receptors they interact with. In vertebrate immune signaling, bacterial sensing modules (for example, TLRs), lipid-binding sorting modules (for example, TIRAP or TRIF) and signaling modules (for example, MyD88 and TRAM) are carried on separate molecules and assemble via transient interactions. Drosophila MyD88 combines sorting and signaling functions in a single molecule, bypassing the need for TIRAP. Notably, rLC merges features of sensing and signaling receptors and sorting adaptors into a single molecule. The fact that Drosophila rLC has no immediate homologs in vertebrates with PGN-sensing and PGN-signaling pathways suggests evolutionary uncoupling of sensing and sorting domains, possibly to increase the spectrum of signaling by combinatorial recruitment of adaptors to sensing receptors (Neyen, 2016).

Accessibility to peptidoglycan is important for the recognition of gram-positive bacteria in Drosophila

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. This study finds that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and PFRP-LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. These results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens (Vaz, 2019).

In Drosophila, the generally accepted explanation for selective activation of immune pathways at the recognition level has been that PGRPs structurally discriminate between different types of PGs. This discrimination occurs on the basis of the amino acid present at position 3 of the stem peptide. Thus, PGRP-SA recognizes Lys (found in Gram-positive bacteria), while PGRP-LC interacts with DAP (found in Gram-negative bacteria and Gram-positive bacilli). The idea of structural discrimination at the level of the stem peptide resulted from a combination of observations, including PG binding assays, structural work, and in vivo infection experiments. However, there are several concerns with the experiments that support this Lys/DAP dichotomy or with the interpretation of the data. A summary of these follows (Vaz, 2019).

First, the PG binding experiments were conducted with a buffer that is often used to solubilize proteins, but that was very different from hemolymph (where interactions between PG and PGRPs take place). Second, the PG used for binding as well as infection experiments was quantified only by weight and not by the number of disaccharide GlcNAc-MurNAC added to the binding reaction. Third, the structural data available and previously described referred to the binding of monomeric PG, whereas PGRP-SA and PGRP-LCx are both able to bind polymeric PG and may do so in vivo. In the context of polymeric PG binding, the small difference of a carboxyl group on the Cε with d-chirality in DAP may not be as important for downstream signaling. Moreover, superposition of the two structures showed that the electrostatic potential at the surface of both PGRPs suggests that binding to both types of PG in solution is probable (Vaz, 2019).

Based on the published structure, the defining characteristic of the binding groove is the conformation of Arg413, which securely anchors DAP-type PG and is likely to impose a preference for the latter via increased conformational stability. This preference was verified with quantitative binding to Lys and DAP-type PG, in which the latter was bound in increased quantities by PGRP-LC. Nevertheless, PGRP-LC did bind Lys-type PG in vitro. Moreover, binding of PGRP-LC to Lys-type bacteria was recorded ex vivo, and this binding resulted in a robust AMP induction that was statistically higher than the one elicited through PGRP-SA, considered the bona fide receptor for Gram-positive bacteria. This indicated that the upstream binding preferences of PGRP-LC did not result in differences in transcriptional AMP induction (Vaz, 2019).

For PGRP-SA, replacement of the anchoring Arg413 from PGRP-LC with a threonine should leave an accommodating void that can flexibly bind via a water molecule network either types of PG. In this case, there should be no noticeable difference in binding affinity. Little difference was found between PGRP-SA binding to Lys PG compared with DAP-type PG (Vaz, 2019).

These results showed that when WTAs were removed, the binding of PGRPs to whole bacteria was significantly improved. This, however, was not reflected in binding assays to purified PG, showing that the increase in binding was about accessibility to PG on the bacterial cell wall, not about changing the WTA-less PG itself. This is consistent with the fact that purified PG from WTA-less bacteria and their parental strains had no significant differences in their HPLC profile analysis that could lead to an improved binding of PGRP-SA or PGRP-LCs (Vaz, 2019).

The above results show that when WTAs were removed from bacteria, the majority of the observed phenotype attributed to selective recognition and downstream signaling based on the bacterial type were either abolished or diminished. This was accompanied by PGRP binding to whole bacteria, irrespective of PG type, in which both PGRPs played a role in host survival and restricting pathogen growth. These results are in conflict with the general consensus of Lys/DAP discrimination and put forward an alternative explanation for differential immune triggering. It is proposed that at least for bacteria that have their PGs exposed to the host environment (Gram-positive bacteria and bacilli), accessibility to PG is a major factor restricting binding, recognition, and downstream signaling. Structural discrimination may still be important when monomeric PG fragments are the only means of 'accessing' PG (in Gram-negative bacteria) (Vaz, 2019).

A fundamental issue when considering pathogen recognition in innate immunity is how a small number of germline coded receptors sense the vast variability of bacterial pathogens. Adapting receptors to an evolutionary conserved bacterial molecule essential for bacterial survival but not present in the host (e.g., PG), has been considered a productive host strategy. However, it is believed that it would have been detrimental for the host to further specialize its non-enzymatic PGRP receptors to PG-stem peptide variants, as this would essentially decrease even more the effective number of possible productive host-pathogen recognition interactions, especially in an animal such as Drosophila, challenged by a wide variety of pathogens, each of which interacts rarely with it. Put it another way, Drosophila is not subject to the arms race-type tight co-evolution with its pathogens that Anopheles is, for example, but it is subject to diffuse co-evolution with an array of non-specialist pathogens (Vaz, 2019).

Evolutionary studies on Drosophila PGRPs (including SA and LC) have shown that there is no strong co-evolution interaction, and as such, no need for specialization. It is proposed that it was the accessibility to PGs on different bacterial cell wall structures that defined how PGRPs were able to bind to PG on whole bacteria and the specified differences in downstream signaling responses, instead of direct structural specialization, as previous studies suggested. Thse results indicate that even when structural preference exists (as in the case of PGRP-LC), differences in downstream signaling are equalized when accessibility to PG on the cell wall is increased. More work is needed to test whether accessibility is also critical for PGRP-mediated recognition of whole bacteria in mammals (Vaz, 2019).

Based on the published structure, the defining characteristic of the binding groove is the conformation of Arg413, which securely anchors DAP-type PG and is likely to impose a preference for the latter via increased conformational stability. This preference was verified with quantitative binding to Lys and DAP-type PG, in which the latter was bound in increased quantities by PGRP-LC. Nevertheless, PGRP-LC did bind Lys-type PG in vitro. Moreover, binding of PGRP-LC to Lys-type bacteria was recorded ex vivo, and this binding resulted in a robust AMP induction that was statistically higher than the one elicited through PGRP-SA, considered the bona fide receptor for Gram-positive bacteria. This indicated that the upstream binding preferences of PGRP-LC did not result in differences in transcriptional AMP induction (Vaz, 2019).

For PGRP-SA, replacement of the anchoring Arg413 from PGRP-LC with a threonine should leave an accommodating void that can flexibly bind via a water molecule network either types of PG. In this case, there should be no noticeable difference in binding affinity. Little difference was found between PGRP-SA binding to Lys PG compared with DAP-type PG (Vaz, 2019).

The results showed that when WTAs were removed, the binding of PGRPs to whole bacteria was significantly improved. This, however, was not reflected in binding assays to purified PG, showing that the increase in binding was about accessibility to PG on the bacterial cell wall, not about changing the WTA-less PG itself. This is consistent with the fact that purified PG from WTA-less bacteria and their parental strains had no significant differences in their HPLC profile analysis that could lead to an improved binding of PGRP-SA or PGRP-LC (Vaz, 2019).

Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium

Barrier epithelia that are persistently exposed to microbes have evolved potent immune tools to eliminate such pathogens. If mechanisms that control Drosophila systemic responses are well-characterized, the epithelial immune responses remain poorly understood. This study consisted of a genetic dissection of the cascades activated during the immune response of the Drosophila airway epithelium i.e. trachea. Evidence is presented that bacteria induced-antimicrobial peptide (AMP) production in the trachea is controlled by two signalling cascades. AMP gene transcription is activated by the inducible IMD pathway that acts non-cell autonomously in trachea. This IMD-dependent AMP activation is antagonized by a constitutively active signalling module involving the receptor Toll-8/Tollo, the ligand Spätzle2/DNT1 (Neurotrophin 1) and Ect-4, the Drosophila ortholog of the human Sterile alpha and HEAT/ARMadillo motif (SARM). The data show that, in addition to Toll-1 whose function is essential during the systemic immune response, Drosophila relies on another Toll family member to control the immune response in the respiratory epithelium (Akhouayri, 2011).

Epithelial responses are local responses to prevent the epithelium from unnecessary immune reactions. Since the recognition steps in Drosophila respiratory epithelia involve the transmembrane receptor PGRP-LC and occur within the extracellular space, it is expected that molecular mechanisms must be at work to prevent constitutive or excessive immune response in this tissue, particularly essential for animal growth and viability. This report presents data demonstrating that the transmembrane receptor Tollo is part of a signalling network, whose function is to specifically down-regulate AMP production in the trachea. Tollo antagonizes IMD pathway activation in the respiratory epithelium, and DNT1/Spz2 and Ect4/SARM are putative Tollo ligand and transducer, respectively, in this process. These data demonstrate that, in addition to the family founder Toll-1, another member of the Leucine-Rich-Repeats family of Toll proteins, is regulating the Drosophila innate immune response. Although it has been abundantly documented that every single mammalian TLR has an immune function, the putative implication of Toll family members, other than Toll-1 itself, in the Drosophila immune response has been a subject of controversy. Data showing that Drosophila Toll-9 over-expression was sufficient to induce AMPs expression in vivo has prompted the idea that Toll-9 could maintain significant levels of anti-microbial molecules, thus providing basal protection against microbes. However, a recent analysis of a complete Toll-9 loss-of-function allele has shown that this receptor is neither implicated in basal anti-microbial response nor required to mount an immune response to bacterial infection (Narbonne-Reveau, 2011. The present data are also fully consistent with a recent report showing that Toll-6, Toll-7 and Toll-8 are not implicated in systemic AMP production in flies, and demonstrate that a Toll family member, Tollo, is a negative regulator of local airway epithelial immune response upon bacterial infection. In contrast to Toll-1, whose activation is inducible in the fat body, Tollo pathway activation seems to be constitutive in the trachea. Despite these differences, both receptors use a member of the Spz family as ligand. Interestingly, sequence similarities, intron's size and conservation of key structural residues, indicate that Spz2/DNT1 is phylogenetically the closest family member to the Toll ligand Spz. Furthermore, both Spz and Spz2/DNT1 have been shown to have neurotrophic functions in flies. It would be of great interest to test whether Tollo also mediates Spz2 function in the nervous system (Akhouayri, 2011).

Both during embryonic development and immune response, Spz is activated by proteolytic cleavage. This step depends upon the Easter protease that is implicated in D/V axis specification and on SPE for Toll pathway activation by microbes. Since Spz orthologs are also produced as longer precursors, they are likely to be activated by proteolysis. The fact that Tollo and Spz2 loss-of-function phenotypes correspond to excessive AMP production, suggests that in wild-type conditions, the Tollo pathway is constitutively activated by an active form of the Spz2 ligand. This situation is reminiscent to that observed in the embryonic ventral follicle cells, in which a Pipe-mediated signal induces a constitutive activation of the Easter cascade leading to Spz cleavage, Toll activation and, in turn, ventral fate acquisition. It should be noted that Easter and one Pipe isoform are very strongly expressed in the trachea cells, and are candidate proteins in mediating Tollo activity in the respiratory epithelia (Akhouayri, 2011).

The fact that Ect4, but not dMyd88 mutant, loss-of-function mutant phenocopies Tollo mutant suggest that Ect4 could be the TIR domain adaptor transducing Tollo signal in the tracheal cells. Alternatively, Ect4/SARM could mediate Tollo function by interfering with IMD pathway signalling. In mammals, SARM is under the transcriptional control of TLR and negatively regulates TLR3 signalling by directly interfering with the association between the RHIM domain-containing proteins TRIF and RIP (Carty, 2006). Since PGRP-LC contains a RHIM domain as TRIF, and IMD is the Drosophila counterpart of RIP, one can envisage that Drosophila SARM could act by interfering with the PGRP-LC/IMD association required for IMD pathway signalling. Similarly to its function as a negative regulator in fly immunity, SARM is the only TIR domain-containing adaptor that acts as a suppressor of TLR signalling (Akhouayri, 2011).

One obvious question relates to the mode of action of Tollo on IMD pathway downregulation. Two mechanisms have been recently described that result in the down-regulation of the IMD pathway. The first one regulates PGRP-LC membrane localization, and is dependent on the PIRK protein (Lhocine, 2008). Upon infection, the intracellular PIRK protein is up-regulated and, in turn, represses PGRP-LC plasma membrane localization leading to the shutdown of the IMD signalling (Lhocine, 2008). In infected pirk mutants, IMD-dependent AMPs are overproduced in both the gut and the fat body. In the conditions used in this study, however, inactivation of PIRK specifically in the trachea did not influence Drosomycin activation in trachea. To verify whether Tollo is acting via a mechanism similar to PIRK, PGRP-LC membrane localization was examined using a UAS-PGRP-LC::GFP construct. PGRP-LC membrane localization was identical in wild-type and Tollo mutant tracheal cells. The second mechanism that modulates IMD activation, acts directly on the promoters of IMD target genes. Caudal transcription factor has been shown to sit on some of the IMD target promoters preventing their activation by Relish. The putative implication of Caudal in Tollo signalling was tested by using Drs-GFP reporter transgenes containing either wild-type Caudal Responsive Elements (CDREs) or mutated versions unresponsive to Caudal activity. Upon infection, Drs-GFP with mutated CDREs was activated in fat body but not in gut or trachea. In conclusion, Caudal acts as a transcriptional activator, rather than a repressor, for the Drs-GFP reporter in trachea. These results indicate that Tollo does not regulate the IMD pathway via PGRP-LC membrane localization or through promoter targeting of Caudal. One challenging task for the future will be to identify the mechanism used by Tollo to counter-balance tracheal PGRP-LC activation. It has been reported that the loss of Tollo function in ectodermal cells during embryogenesis alters glycosylation in nearby differentiating neurons. Since the pattern of oligosaccharides expressed in a cell can influence its interactions with others and with pathogens, Tollo could function by modifying glycosylation pattern in response to microbes. It could be envisaged that Tollo mediates PGRP-LC glycosylation, and thereby reduces its ability to respond to bacterial elicitors. Further work will be required to address the above hypothesis, whereby Tollo activity and glycosylation modification could be linked in order to regulate the IMD pathway activation in trachea (Akhouayri, 2011).

Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling

Caspases have been extensively studied as critical initiators and executioners of cell death pathways. However, caspases also take part in non-apoptotic signalling events such as the regulation of innate immunity and activation of nuclear factor-κB (NF-κB). How caspases are activated under these conditions and process a selective set of substrates to allow NF-κB signalling without killing the cell remains largely unknown. This study shows that stimulation of the Drosophila pattern recognition protein PGRP-LCx induces DIAP2-dependent polyubiquitylation of the initiator caspase DREDD. Signal-dependent ubiquitylation of DREDD is required for full processing of IMD, NF-κB/Relish and expression of antimicrobial peptide genes in response to infection with Gram-negative bacteria. The results identify a mechanism that positively controls NF-κB signalling via ubiquitin-mediated activation of DREDD. The direct involvement of ubiquitylation in caspase activation represents a novel mechanism for non-apoptotic caspase-mediated signalling (Meinander, 2012).

Cleavage of PGRP-LC receptor in the Drosophila IMD pathway in response to live bacterial infection in S2 cells

Drosophila responds to Gram-negative bacterial infection by activating the immune deficiency (IMD) pathway, leading to production of antimicrobial peptides (AMPs). As a receptor for the IMD pathway, peptidoglycan-recognition protein (PGRP), PGRP-LC is known to recognize and bind monomeric peptidoglycan (DAP-type PGN) through its PGRP ectodomain and in turn activate the IMD pathway. The questions remain how PGRP-LC is activated in response to pathogen infection to initiate the IMD signal transduction in Drosophila. This study presents evidence to show that proteases such as elastase and Mmp2 can also activate the IMD pathway but not the TOLL pathway. The elastase-dependent IMD activation requires the receptor PGRP-LC. Importantly, it was found that live Salmonella/E. coli infection modulates PGRP-LC expression/receptor integrity and activates the IMD pathway while dead Salmonella/E. coli or protease-deficient E. coli do neither. These results suggest an interesting possibility that Gram-negative pathogen infection may be partially monitored through the structural integrity of the receptor PGRP-LC via an infection-induced enzyme-based cleavage-mediated activation mechanism (Schmidt, 2011).

Infection-induced proteolysis of PGRP-LC controls the IMD activation and melanization cascades in Drosophila

The Drosophila immune deficiency (IMD) pathway, homologous to the mammalian tumor necrosis factor (TNF-alpha) signaling pathway, initiates antimicrobial peptide (AMP) production in response to infection by gram-negative bacteria. A membrane-spanning peptidoglycan recognition protein, PGRP-LC, functions as the receptor for the IMD pathway. This receptor is activated via pattern recognition and binding of monomeric peptidoglycan (DAP-type PGN) through the PGRP ectodomain. This article shows that the receptor PGRP-LC is down-regulated in response to Salmonella/Escherichia coli infection but is not affected by Staphylococcus infection in vivo, and an ectodomain-deleted PGRP-LC lacking the PGRP domain is an active receptor. The receptor PGRP-LC regulates and integrates two host defense systems: the AMP production and melanization. A working model is proposed in which pathogen invasion and tissue damage may be monitored through the receptor integrity of PGRP-LC after host and pathogen are engaged via pattern recognition. The irreversible cleavage or down-regulation of PGRP-LC may provide an additional cue for the host to distinguish pathogenic microbes from nonpathogenic ones and to subsequently activate multiple host defense systems in Drosophila, thereby effectively combating bacterial infection and initiating tissue repair (Schmidt, 2008).

Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites

Recognition of peptidoglycan (PGN) is paramount for insect antibacterial defenses. In the fruit fly Drosophila melanogaster, the transmembrane PGN Recognition Protein LC (PGRP-LC) is a receptor of the Imd signaling pathway that is activated after infection with bacteria, mainly Gram-negative (Gram-). This study demonstrates that bacterial infections of the malaria mosquito Anopheles gambiae are sensed by the orthologous PGRPLC protein which then activates a signaling pathway that involves the Rel/NF-kappaB transcription factor REL2. PGRPLC signaling leads to transcriptional induction of antimicrobial peptides at early stages of hemolymph infections with the Gram-positive (Gram+) bacterium Staphylococcus aureus, but a different signaling pathway might be used in infections with the Gram- bacterium Escherichia coli. The size of mosquito symbiotic bacteria populations and their dramatic proliferation after a bloodmeal, as well as intestinal bacterial infections, are also controlled by PGRPLC signaling. This defense response modulates mosquito infection intensities with malaria parasites, both the rodent model parasite, Plasmodium berghei, and field isolates of the human parasite, Plasmodium falciparum. It is proposed that the tripartite interaction between mosquito microbial communities, PGRPLC-mediated antibacterial defense and infections with Plasmodium can be exploited in future interventions aiming to control malaria transmission. Molecular analysis and structural modeling provided mechanistic insights for the function of PGRPLC. Alternative splicing of PGRPLC transcripts produces three main isoforms, of which PGRPLC3 appears to have a key role in the resistance to bacteria and modulation of Plasmodium infections. Structural modeling indicates that PGRPLC3 is capable of binding monomeric PGN muropeptides but unable to initiate dimerization with other isoforms. A dual role of this isoform is hypothesized: it sequesters monomeric PGN dampening weak signals and locks other PGRPLC isoforms in binary immunostimulatory complexes further enhancing strong signals (Meister, 2009).

Pirk is a negative regulator of the Drosophila Imd pathway

NF-kappaB transcription factors are involved in evolutionarily conserved signaling pathways controlling multiple cellular processes including apoptosis and immune and inflammatory responses. Immune response of the fruit fly Drosophila melanogaster to Gram-negative bacteria is primarily mediated via the Imd (immune deficiency) pathway, which closely resembles the mammalian TNFR signaling pathway. Instead of cytokines, the main outcome of Imd signaling is the production of antimicrobial peptides. The pathway activity is delicately regulated. Although many of the Imd pathway components are known, the mechanisms of negative regulation are more elusive. This study reports that a previously uncharacterized gene, pirk, is highly induced upon Gram-negative bacterial infection in Drosophila in vitro and in vivo. pirk encodes a cytoplasmic protein that coimmunoprecipitates with Imd and the cytoplasmic tail of peptidoglycan recognition protein LC (PGRP-LC). RNA interference-mediated down-regulation of Pirk caused Imd pathway hyperactivation upon infection with Gram-negative bacteria, while overexpression of pirk reduced the Imd pathway response both in vitro and in vivo. Furthermore, pirk-overexpressing flies were more susceptible to Gram-negative bacterial infection than wild-type flies. It is concluded that Pirk is a negative regulator of the Imd pathway (Kleino, 2008).

Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway

Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, this study identified Rudra (also termed Pirk), a new regulator of the IMD pathway, and demonstrates that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression is rapidly induced. In cells, RNAi targeting of rudra causes a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and are more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors (Aggarwal, 2008).

PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling

Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. This study reports that a recently identified Drosophila immune regulator, which is termed PGRP-LC-interacting inhibitor of Imd signaling (PIMS, also termed Pirk), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections (Lhocine, 2008).

The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation

Eukaryotic peptidoglycan recognition proteins (PGRPs) are related to bacterial amidases. In Drosophila, PGRPs bind peptidoglycan and function as central sensors and regulators of the innate immune response. PGRP-LC/PGRP-LE constitute the receptor complex in the immune deficiency (IMD) pathway, which is an innate immune cascade triggered upon Gram-negative bacterial infection. This study presents the functional analysis of the nonamidase, membrane-associated PGRP-LF. PGRP-LF acts as a specific negative regulator of the IMD pathway. Reduction of PGRP-LF levels, in the absence of infection, is sufficient to trigger IMD pathway activation. Furthermore, normal development is impaired in the absence of functional PGRP-LF, a phenotype mediated by the JNK pathway. Thus, PGRP-LF prevents constitutive activation of both the JNK and the IMD pathways. A model is proposed in which PGRP-LF keeps the Drosophila IMD pathway silent by sequestering circulating peptidoglycan (Maillet, 2008).

Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster

The inducible expression of antimicrobial peptide genes in Drosophila melanogaster is regulated by the conserved Toll and peptidoglycan recognition protein LC/immune deficiency (PGRP-LC/IMD) signaling pathways. It has been proposed that the two pathways have independent functions and mediate the specificity of innate immune responses towards different microorganisms. Scattered evidence also suggests that some antimicrobial target genes can be activated by both Toll and IMD, albeit to different extents. This dual activation can be mediated by independent stimulation or by cross-regulation of the two pathways. This report shows that the Toll and IMD pathways can interact synergistically, demonstrating that cross-regulation occurs. The presence of Spatzle (the Toll ligand) and gram-negative peptidoglycan (the PGRP-LC ligand) together causes synergistic activation of representative target genes of the two pathways, including Drosomycin, Diptericin, and AttacinA. Constitutive activation of Toll and PGRP-LC/IMD can mimic the synergistic stimulation. RNA interference assays and promoter analyses demonstrate that cooperation of different NF-kappaB-related transcription factors mediates the synergy. These results illustrate how specific ligand binding by separate upstream pattern recognition receptors can be translated into a broad-spectrum host response, a hallmark of innate immunity (Tanji, 2007).

The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila

From a forward genetic screen for phagocytosis mutants in Drosophila, a mutation was identified that affects peptidoglycan recognition protein (PGRP) SC1a and impairs the ability to phagocytose the bacteria Staphylococcus aureus, but not Escherichia coli and Bacillus subtilis. Because of the differences in peptidoglycan peptide linkages in these bacteria, the data suggest that the Drosophila gene PGRP-SC1a is necessary for recognition of the Lys-type peptidoglycan typical of most Gram⁺ bacteria. PGRP-SC1a mutants also fail to activate the Toll/NF-kappaB signaling pathway and are compromised for survival after S. aureus infection. This mutant phenotype is the first found for an N-acetylmuramoyl-L-alanine amidase PGRP that cleaves peptidoglycan at the lactylamide bond between the glycan backbone and the crosslinking stem peptides. By generating transgenic rescue flies that express either wild-type or a noncatalytic cysteine-serine mutant PGRP-SC1a, it was found that PGRP-SC1a amidase activity is not necessary for Toll signaling, but is essential for uptake of S. aureus into the host phagocytes and for survival after S. aureus infection. Furthermore, the PGRP-SC1a amidase activity can be substituted by exogenous addition of free peptidoglycan, suggesting that the presence of peptidoglycan cleavage products is more important than the generation of cleaved peptidoglycan on the bacterial surface for PGRP-SC1a mediated phagocytosis (Garver, 2006).

Host receptors must recognize a microbe before any immune response can begin. After recognition, signaling pathways are activated and result in effector responses such as induction of antimicrobial peptides (AMPs), melanization, and phagocytosis, which are important for controlling the infection. Much is known about the signaling pathways important for the AMP response in Drosophila, but less is known about the regulation of the other two responses. In Drosophila, if phagocytosis is blocked in a mutant that is already impaired for the AMP response, a normally innocuous Escherichia coli infection becomes lethal. Phagocytosis is also likely to be a first line of defense, because the response involves specific interaction between microbe and host receptors and occurs within half an hour, whereas the induction of AMPs occurs over several hours and AMPs can act on diverse groups of microbes (Garver, 2006).

The peptidoglycan recognition proteins (PGRPs) are critical receptors in the Drosophila immune response that are required for the recognition of peptidoglycan, a component of bacterial cell walls (Dziarski, 2004; Steiner, 2004), and for subsequent activation of AMP gene expression (Choe, 2002; Werner, 2003). PGRPs were first characterized in the moths Bombyx mori and Trichoplusia ni and proposed to be receptors that can trigger immune responses. PGRPs have also been identified in mammals, and mutant PGRP mice have been generated, but the most comprehensive characterization of PGRPs has been performed in Drosophila (Garver, 2006).

Drosophila has 13 PGRP genes, six long (L) forms with four that are predicted to reside in the plasma membrane, and seven short forms (S) that are all predicted to be secreted. PGRPs share homology with N-acetylmuramoyl-L-alanine amidases, which cleave peptidoglycan at the lactylamide bond between the glycan backbone and the stem peptides. Some PGRPs, such as PGRP-LC, -LE -SA, and -SD, lack a critical cysteine in the catalytic pocket and are not able to cleave peptidoglycan. PGRP-LC, -LE, and -SA have been demonstrated to bind peptidoglycan and are necessary for expression of AMP genes, supporting the hypothesis that PGRPs directly recognize bacteria and activate immune responses. The identification of mutations in PGRP-SA (seml) and PGRP-LC (ird7 or totem) indicated that these genes are necessary for activation of the two signaling pathways regulating AMP gene expression, the Toll pathway that responds to Gram⁺ bacteria and fungi and the Imd pathway that responds to Gram^– bacteria. Drosophila uses PGRP-SA and PGRP-LC to distinguish between Gram⁺ and Gram^– PGN for activation of the Toll and Imd signaling pathways (Leulier, 2003). Gram⁺ PGN and Gram^– PGN differ in the stem peptide portion; typical Gram⁺ bacteria have a lysine as the third amino acid, whereas Gram^– bacteria and the Gram⁺ Bacillus have a diaminopimelic acid (DAP) in that position. Two other noncatalytic PGRPs, PGRP-LE and PGRP-SD, also play a role in activation of the Imd and Toll pathways, respectively. PGRP-LC, PGRP-LE double mutants show a more dramatic phenotype to Bacillus and Gram^– bacterial infection than either mutation alone, suggesting that PGRP-LE acts with PGRP-LC in the recognition of Gram^– DAP-type peptidoglycan for the activation of the Imd pathway. PGRP-SD may be playing a similar role with PGRP-SA for activation of the Gram⁺/Toll pathway (Garver, 2006 and references therein).

Catalytic PGRPs, such as PGRP-SC1a and -SC1b, include this cysteine residue in the active site, and are potent enzymes that cleave peptidoglycan between the N-acetylmuramic acid of the backbone and the L-alanine in the stem peptide. After digestion with PGRP-SC1b, staphylococcal peptidoglycan exhibits less activation of the AMP genes in a Drosophila blood cell line, so it was hypothesized that catalytic PGRPs may act as scavengers to limit an inflammatory response to free peptidoglycan (Mellroth, 2003). This may not be absolute, since PGRP-SC1b-digested Gram^– peptidoglycan is still able to activate the AMP response in S2 cells, albeit at a higher dose. However, it was of interest to examine the role of these genes in vivo (Garver, 2006).

In a genetic screen for phagocytosis mutants, a novel mutant, picky was identified. picky flies fail to phagocytose S. aureus particles but can phagocytose E. coli, Bacillus subtilis, and Saccharomyces cerevisiae zymosan particles. picky mutants have defects in the activation of the Toll pathway. picky maps to the location of the PGRP-SC1a, -SC1b, and -SC2 genes, and picky flies express significantly less PGRP-SC1a. The picky defects can be rescued by expression of PGRP-SC1a, indicating that PGRP-SC1a is important for phagocytosis and activation of AMP responses. These results differ from the prevailing model of catalytic PGRPs as scavengers and suggest that in vivo, PGRP-SC1a is required for initiating immune pathways. By comparing a wild-type PGRP-SC1a with a cysteine-serine mutant PGRP-SC1a for rescue of picky phenotypes, it was found that the catalytic activity is essential for phagocytosis of live S. aureus, but not for activation of the Toll pathway (Garver, 2006).

To identify genes important for phagocytosis, a collection of ethylmethane sulfonate-induced adult viable Drosophila mutants was screened using an in vivo phagocytosis assay. To determine what a mutant might look like, the PGRP-LC (ird7) and PGRP-SA (seml) mutants were examined. PGRP-LC has been reported to be a recognition receptor for phagocytosis of E. coli in S2 cells. However, this study contradicted another report citing that ird7 blood cells can phagocytose bacteria (Choe, 2002). The current study found that ird7 mutants are able to phagocytose both E. coli and S. aureus particles. This finding suggested that phagocytosis of Gram^– bacteria may require other receptors in addition to PGRP-LC in vivo. In contrast, it was found that seml mutants are specifically impaired in their ability to phagocytose S. aureus but not E. coli. 94% of the seml mutant flies were able to phagocytose E. coli, but only 25% were able to phagocytose S. aureus. This finding suggests that PGRP-SA may be important for recognition of Gram⁺ bacteria for phagocytosis in addition to its role in activating the Toll pathway. In contrast, flies with mutations affecting other Toll pathway components, spatzle (the Toll ligand) and Dif (an NF-kappaB) were still able to phagocytose S. aureus, indicating that the phagocytosis defect is not a secondary effect from loss of Toll signaling (Garver, 2006).

From a pilot screen, one mutation was identified that was named picky eater^Z2–4761 (picky) because the mutants fail to phagocytose S. aureus particles, but can still phagocytose E. coli and Saccharomyces cerevisiae zymosan particles. Only 25% of picky mutant flies showed any phagocytosis response to S. aureus, but 83% of those same flies were still able to phagocytose E. coli. To investigate whether the recognition involved the difference in peptidoglycan between Gram⁺ and Gram^– bacteria, phagocytosis was examined of a B. subtilis-GFP strain. picky mutants are able to phagocytose B. subtilis. This finding suggests that the picky gene product is important for the specific recognition of the Lys-type peptidoglycans, typical of most Gram⁺ bacteria, but not found in Bacillus spp (Garver, 2006).

Because picky flies appear to be impaired in the recognition of S. aureus for phagocytosis and survival, it was possible that the mutation might also affect activation of the Toll or Imd pathways. The induction of Drosomycin and Diptericin AMP genes is often used to assess activation of the Toll/Gram⁺ and Imd/Gram^– signaling pathways, respectively. The AMP responses in picky were compared with those of the known Toll pathway components, seml and Dif, and to an Imd pathway component, ird7. picky flies showed no induction of Drosomycin in response to S. aureus, E. coli, or Micrococcus luteus at either 2 or 24 h. All of the S. aureus-infected picky flies were dead at 24 h, so the Drosomycin expression in this sample was not assessed. seml and Dif both show some induction of Drosomycin at 2 and 24 h. In comparison, picky has a more consistent and dramatic effect on Drosomycin expression and likely encodes an essential component of the Toll pathway. In contrast, the expression of Diptericin in response to bacterial infection in picky mutants was higher than that seen in wild type. Therefore, the defect in picky appears to be selective for the Toll pathway. To determine where in the Toll pathway picky might lie, the picky mutation was crossed into a Toll^10b gain of function mutant. picky was not able to suppress the constitutive expression of Drosomycin in a Toll^10b mutant. This epistasis analysis places picky either upstream of Toll, which would be consistent with a role in bacterial recognition or in a parallel pathway (Garver, 2006).

The peptidoglycan recognition protein family is important for allowing the Drosophila immune response to distinguish between Gram⁺ and Gram^– bacteria for the activation of specific AMP signaling pathways (Choe, 2002; Werner, 2003). The current data indicate that phagocytosis also relies on PGRPs to distinguish between Gram⁺ and Gram^– bacterial peptidoglycan. A catalytic PGRP, PGRP-SC1a, is essential for the phagocytosis of S. aureus and the activation of AMP responses. The fact that PGRP-SC1a mutants have a striking phagocytosis defect indicates that it is absolutely required and that other PGRPs are not able to substitute for its loss of function. PGRP-SC1a differs from the existing PGRP mutants in that it has catalytic activity that is essential for efficient phagocytosis and ultimately limiting the infection process. Hence, noncatalytic PGRPs, such as PGRP-SA, may not be able to substitute for PGRP-SC1a function. Because PGRP-SC1a is likely present in the hemolymph, it may be acting as an opsonin to bind bacteria, with the PGRP-bacterial complex afterwards being recognized by transmembrane phagocytosis receptors that complete the uptake into the phagocyte. An alternative model is that PGRP-SC1a may generate peptidoglycan cleavage products that function as immune modulators to stimulate phagocytic activity (Garver, 2006).

For S. aureus, PGRP-SC1a is necessary for recognition of bacteria for both the phagocytosis and AMP responses. However, for E. coli, PGRP-SC1a is similar to PGRP-LC in that it is not necessary for recognition for phagocytosis, but is necessary for the activation of an AMP response. This finding raises the interesting issue of the role PGRPs play in recognizing Gram^– bacteria. In Gram^– bacteria, the peptidoglycan is buried under an outer membrane, so it has been proposed that the small amounts of PGN that are shed may be sufficient to trigger the AMP response (Leulier, 2003). Because the cell wall PGN is not easily accessible, it makes sense that the PGRPs are not playing a direct role in recognition for phagocytosis. An alternate possibility is that the exposure of bacterial peptidoglycan occurs after the initial uptake into phagocytes, and at that later point, is then recognized by PGRP-LC and PGRP-SC1a to trigger AMP responses. There is precedence for intracellular recognition in the phagosome in mammalian immune responses, with studies indicating that mammalian short PGRPs function in the phagosome to inhibit bacterial growth and the observation that Toll-like receptor recognition of bacteria in the phagosome can influence the rate of phagosome maturation. A related possibility is that some basal level of activation of PGRP-SC1a and the Toll pathway may be required for any expression of Drosomycin. picky mutants show much lower expression of Drosomycin than wild type in the absence of infection, so perhaps an induction may not be detectable (Garver, 2006).

It was also found that seml mutants are defective in the phagocytosis of S. aureus but not E. coli. Recent reports have indicated that the processing of peptidoglycan into monomers, lactyltetrapeptides, or muropeptides, can yield potent activators of the AMP response for both the Imd and the Toll pathway. It was further suggested for the Toll pathway that processing of peptidoglycan may be a prerequisite step upstream of PGRP-SA/seml recognition and activation of the Toll AMP pathway. This model would be consistent with catalytic PGRPs, such as PGRP-SC1a, playing a supporting role in the processing of peptidoglycan for initiating recognition events (Garver, 2006).

Since many PGRPs are clearly required for activation of immune effector responses, the issue arises as to the specificity of PGRPs for their substrates. Work from several groups indicates that alternative splice variants (in PGRP-LC) or small differences in amino acid sequences (from PGRP-LB and human PGRP-Ialpha structural analyses) may result in the ability of different PGRPs to have distinct recognition potential. It will be interesting to explore the range of bacterial types recognized by the 17 Drosophila PGRP proteins and to determine how subtle differences in recognition may be reflected by specific amino acid sequences. There may also be genetic interactions or antagonism in their function, so using the genetic tools available in Drosophila may be necessary for ultimately determining both their unique and redundant roles (Garver, 2006).

This work demonstrates the utility of a forward genetic approach to understanding the recognition of pathogen types for phagocytosis and activation of AMP responses. It is hoped that identification of genes important for recognition of bacteria in Drosophila should increase understanding of how the process works in the immune system (Garver, 2006).

Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor

Tracheal cytotoxin (TCT), a naturally occurring fragment of Gram-negative peptidoglycan, is a potent elicitor of innate immune responses in Drosophila. It induces the heterodimerization of its recognition receptors, the peptidoglycan recognition proteins (PGRPs) LCa and LCx, which activates the immune deficiency pathway. The crystal structure at 2.1 angstrom resolution of TCT in complex with the ectodomains of PGRP-LCa and PGRP-LCx shows that TCT is bound to and presented by the LCx ectodomain for recognition by the LCa ectodomain; the latter lacks a canonical peptidoglycan-docking groove conserved in other PGRPs. The interface, revealed in atomic detail, between TCT and the receptor complex highlights the importance of the anhydro-containing disaccharide in bridging the two ectodomains together and the critical role of diaminopimelic acid as the specificity determinant for PGRP interaction (Chang, 2006).

Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. This study determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. It was shown that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity (Chang, 2005).

Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro

Drosophila knockout mutants have placed peptidoglycan recognition proteins (PGRPs) in the two major pathways controlling immune gene expression. This study examined PGRP affinities for peptidoglycan. PGRP-SA and PGRP-LCx are bona fide pattern recognition receptors, and PGRP-SA, the peptidoglycan receptor of the Toll/Dif pathway, has selective affinity for different peptidoglycans. PGRP-LCx, the default peptidoglycan receptor of the Imd/Relish pathway, has strong affinity for all polymeric peptidoglycans tested and for monomeric peptidoglycan. PGRP-LCa does not have affinity for polymeric or monomeric peptidoglycan. Instead, PGRP-LCa can form heterodimers with LCx when the latter is bound to monomeric peptidoglycan. Hence, PGRP-LCa can be said to function as an adaptor, thus adding a new function to a member of the PGRP family (Mellroth, 2005).

Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor

Drosophila peptidoglycan recognition protein LC (PGRP-LC), a transmembrane protein required for the response to bacterial infection, acts at the top of a cytoplasmic signaling cascade that requires the death-domain protein Imd and an IkappaB kinase to activate Relish, an NF-kappaB family member. It is not clear how binding of peptidoglycan to the extracellular domain of PGRP-LC activates intracellular signaling because its cytoplasmic domain has no homology to characterized proteins. This study demonstrates that PGRP-LC binds Imd and that its cytoplasmic domain is critical for its activity, suggesting that PGRP-LC acts as a signal-transducing receptor. The PGRP-LC cytoplasmic domain is also essential for the formation of dimers, and results suggest that dimerization may be required for receptor activation. The PGRP-LC cytoplasmic domain can mediate formation of heterodimers between different PGRP-LC isoforms, thereby potentially expanding the diversity of ligands that can be recognized by the receptor (Choe, 2005).

The data presented in this study demonstrate that PGRP-LC is an essential component of a signal-transducing receptor complex that presumably binds peptidoglycan PAMPs and initiates the cellular response to this PAMP. The other PGRP required in a Drosophila immune response, PGRP-SA, is a small, soluble protein that circulates in the hemolymph and is required for activation of a hemolymph protease cascade. In contrast, PGRP-LC is a transmembrane protein that acts as a PRR that directly couples pathogen recognition to intracellular signaling (Choe, 2005).

All isoforms of PGRP-LC share a common cytoplasmic domain that has no similarity to other proteins. Nevertheless, the deletion analysis presented in this study defines three functions for the previously uncharacterized cytoplasmic domain of PGRP-LC. First, the membrane-proximal half of the PGRP-LC cytoplasmic domain is required for formation of multimeric receptor complexes. Interactions between PGRP-LC isoforms have been detected, but it was not known which domains mediated interaction. As noted previously, the ability to form heteromeric complexes may broaden the spectrum of PAMPs that can be recognized. Second, PGRP-LC can form a complex with the cytoplasmic protein Imd. The current experiments demonstrate that the N-terminal domain of Imd is essential for its interaction with PGRP-LC, although that interaction need not be direct. Third, the PGRP-LC cytoplasmic domain is necessary and sufficient for activation of downstream signaling, as assayed by expression of CecA1. The results suggest that activation of signaling could involve the following sequence of events: binding of bacterial proteoglycan to PGRP-LC induces receptor multimerization (which can be mimicked by overexpression); and multimerization of the receptor activates the protein complex that includes both PGRP-LC and Imd, ultimately leading to activation of the IkappaB kinase (Ird5) and Relish activation (Choe, 2005).

Although PGRP-LC has no homology to TLRs, the data highlight parallels between the PGRP-LC/Imd and TLR pathways. TLR signaling requires a membrane complex that includes the TLR and death-domain protein MyD88; similarly, signaling from PGRP-LC occurs in a complex that includes the death-domain protein Imd. TLR activity causes activation of an IkappaB kinase complex that phosphorylates the inhibitor protein IkappaB (32); similarly, activation of PGRP-LC leads to activation of an IkappaB kinase complex that phosphorylates the Rel-Ank protein Relish. The adaptor molecule MyD88 has an N-terminal death domain and the C-terminal TIR domain. The MyD88 TIR domain binds the TIR domain of TLR and the MyD88 death domain binds the death domain of the kinase IRAK, thereby linking the membrane receptor to downstream events. Although the cytoplasmic domain of PGRP-LC is not similar to any other protein, these data suggest that it is required for interaction with the previously uncharacterized N-terminal domain of Imd. The C-terminal half (delta132-273) of Imd contains a death domain and can interact with another death-domain protein, dFADD, which is required for downstream signaling and could be also a part of the receptor complex (Choe, 2005).

In animals, the immune-responsive tissues are the fat body, blood cells, and epidermis. Although the interactions described described in this study occur in Schneider cells, a hemocyte line, it is likely that PGRP-LC and Imd interact in the fat body and possibly the epidermis as well. Imd and PGRP-LC mutants have very similar effects on the antimicrobial peptide response in flies, and most of the antimicrobial peptides are produced in the fat body. Overexpression of PGRP-LC in the fat body is sufficient to drive antimicrobial peptide induction, indicating that the fat-body cells are ready to initiate the signaling upon the presence of active PGRP-LC. The response to septic wounding that was used to define the phenotypes of both Imd and PGRP-LC mutants can occur in the absence of blood cells. Imd also acts in the epidermis to allow a local response to septic wounding, and it is likely that PGRP-LC also acts at that site. Although this study provides information about the biochemical mechanism of action of PGRP-LC, additional work will be required to define the diversity of functions of PGRP-LC in systemic immune responses (Choe, 2005).

PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan

Drosophila rely entirely on an innate immune response to combat microbial infection. Diaminopimelic acid-containing peptidoglycan, produced by Gram-negative bacteria, is recognized by two receptors, PGRP-LC and PGRP-LE, and activates a homolog of transcription factor NF-kappaB through the Imd signaling pathway. This study shows that full-length PGRP-LE acts as an intracellular receptor for monomeric peptidoglycan, whereas a version of PGRP-LE containing only the PGRP domain functions extracellularly, like the mammalian CD14 molecule, to enhance PGRP-LC-mediated peptidoglycan recognition on the cell surface. Interaction with the imd signaling protein was not required for PGRP-LC signaling. Instead, PGRP-LC and PGRP-LE signaled through a receptor-interacting protein homotypic interaction motif-like motif. These data demonstrate that like mammals, Drosophila use both extracellular and intracellular receptors, which have conserved signaling mechanisms, for innate immune recognition (Kaneko, 2006).

Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway

Insects depend solely upon innate immune responses to survive infection. These responses include the activation of extracellular protease cascades, leading to melanization and clotting, and intracellular signal transduction pathways inducing antimicrobial peptide gene expression. In Drosophila, the IMD pathway is required for antimicrobial gene expression in response to gram-negative bacteria. The exact molecular component(s) from these bacteria that activate the IMD pathway remain controversial. This study found that highly purified LPS did not stimulate the IMD pathway. However, lipid A, the active portion of LPS in mammals, activated melanization in the silkworm Bombyx morii. On the other hand, the IMD pathway was remarkably sensitive to polymeric and monomeric gram-negative peptidoglycan. Recognition of peptidoglycan required the stem-peptide sequence specific to gram-negative peptidoglycan and the receptor PGRP-LC. Recognition of monomeric and polymeric peptidoglycan required different PGRP-LC splice isoforms, while lipid A recognition required an unidentified soluble factor in the hemolymph of Bombyx morii (Kaneko, 2004).

Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. This study generated PGRP-LE null mutants and reports that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested (Takehana, 2004).

Functional diversity of the Drosophila PGRP-LC gene cluster in the response to lipopolysaccharide and peptidoglycan

The peptidoglycan recognition protein PGRP-LC is a major activator of the imd/Relish pathway in the Drosophila immune response. Three transcripts are generated by alternative splicing of the complex PGRP-LC gene. The encoded transmembrane proteins share an identical intracellular part, but each has a separate extracellular PGRP-domain: x, y, or a. This study shows that two of these isoforms play unique roles in the response to different microorganisms. Using RNA interference in Drosophila mbn-2 cells, it was found that PGRP-LCx is the only isoform required to mediate signals from Gram-positive bacteria and purified bacterial peptidoglycan. By contrast, the recognition of Gram-negative bacteria and bacterial lipopolysaccharide requires both PGRP-LCa and LCx. The third isoform, LCy, is expressed at lower levels and may be partially redundant. Two additional PGRP domains in the gene cluster, z and w, are both included in a single transcript of a separate gene, PGRP-LF. Suppression of this transcript does not block the response to any of the microorganisms tested (Werner, 2003).

Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-kappaB precursors. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Ikappa-B kinase complex (dmIKK). dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP). Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway. Using inducible expression of dFADD double-stranded RNA, this study demonstrates that dFADD is a novel component of the Imd pathway: dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. These results reinforce the parallels between the Imd and the TNF-R1 pathways (Leulier, 2002).

Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila

Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. This study shows that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-kappaB family transcription factor Relish (Choe, 2002).

In response to infection,Drosophila activates the transcription of a battery of antimicrobial peptide genes in cells of the fat body (the insect analog of the liver). Two major branches of this humoral response have been identified: as in mammals, these responses require NF-kappaB transcription factors. One branch activates antifungal responses and requires the receptor Toll and the NF-kappaB family transcription factor Dif. The second branch, which is primarily antibacterial, requires the NF-kappaB protein Relish, an IkappaB kinase (IKK), a caspase, a mitogen-activated protein kinase kinase kinase, and the death-domain protein Imd (Choe, 2002).

This study has taken a genetic approach to identifying genes required for the antibacterial response. One gene that is absolutely required for the induction of the antibacterial response is ird7 (immune response deficient 7). Two mutations in ird7 identified in an ethylmethane sulfonate (EMS) mutagenesis screen prevented the induction of three antibacterial peptide genes, Diptericin,Cecropin, and Defensin, after infection by either Gram-negative or Gram-positive bacteria. Three other antimicrobial peptide genes, Attacin, Metchnikowin, andDrosomycin, also failed to be induced to normal levels. The profile of antimicrobial gene expression observed in theird7 mutants was similar to that observed in imd,DmIkkβ/ird5, and Relish mutants after bacterial infection, but was distinct from that of Toll andDif mutants. This pattern suggests thatird7 is an essential component of the same signaling pathway that requires imd and Relish, but is not required for the Toll-Dif pathway. Both ird7 mutants are homozygous viable and fertile, and blood cells from ird7 mutants can phagocytose bacteria; these findings suggest thatird7 is required specifically for the humoral immune response (Choe, 2002).

The transcription factor Relish directly activates antibacterial target genes in Drosophila. Relish is a compound protein similar to mammalian p100 and p105 (the precursors of the p52 and p50 subunits of NF-kappaB), with an NH₂-terminal Rel homology and a COOH-terminal ankyrin repeat domain similar to that of the NF-kappaB inhibitor IkappaB. In response to immune challenge, full-length Relish (REL-110) is endoproteolytically clipped to generate the NH₂-terminal REL-68 fragment, which translocates into the nucleus, and the COOH-terminal REL-49 ankyrin repeat fragment, which remains stable in the cytoplasm. In contrast to wild-type animals, no processing of Relish was detected in ird7 mutant larva. The Rel domain of Relish failed to translocate to fat body nuclei in ird7 mutants. These results indicate that ird7 is required for Relish processing and nuclear translocation (Choe, 2002).

Recombination and deficiency mapping localized ird7 to a small interval on the third chromosome, 66F5-67A9 . The Drosophila genome sequence annotation indicates the presence of 12 genes in this region, including two genes encoding peptidoglycan recognition protein (PGRP) domains, PGRP-LA and PGRP-LC. Peptidoglycan is a strong activator of innate immune responses in insects and mammals, and a PGRP was first identified in a silk moth (Bombyx) on the basis of its ability to bind peptidoglycan and activate one aspect of the immune response, the prophenoloxidase cascade. Later studies have implicated PGRPs in innate immune responses from arthropods to mammals (Choe, 2002).

This study identified sequence changes that would disrupt the function ofPGRP-LC in both ird7 alleles. The gene was represented by several expressed sequence tag clones that encode a single splice form, designated PGRP-LCa. In addition, sequences encoding two additional exons encoding PGRP domains ('x' and 'y') were identified in an intron of PGRP-LC. A screen of a larval-pupal cDNA library was performed with the x and y exons, and an alternatively spliced form of PGRP-LC was identified that included the x exon; this isoform was called PGRP-LCx. Both PGRP-LC isoforms encoded type II transmembrane proteins with common NH₂-terminal cytoplasmic and transmembrane domains but different extracellular domains. The extracellular PGRP domains of the two isoforms were only 38% identical (55 of 145 residues). Northern hybridization with a common PGRP-LC exon probe revealed transcripts about 2.0 kb in size in wild-type larvae, but no transcript of that size in ird7¹ mutant animals; instead, a larger transcript of lower abundance was detected. Sequence analysis revealed an insertion of 858 base pairs (bp) of single-copy sequence into exon 2, which is the first coding exon in both isoforms, in the ird7¹ allele. This insertion introduced a stop codon and would generate a truncated cytoplasmic protein. No sequence change in the PGRP-LCa isoform was identified in the ird7² allele. However, there was a G to A substitution in the x PGRP domain in thePGRP-LCx isoform of ird7² , which introduced a stop codon that makes a truncated protein lacking the last 107 amino acids of this isoform. Because the ird7² allele alters only PGRP-LCx and has a profound effect on antimicrobial gene expression, this isoform must play a crucial role in vivo. The specific requirement for the PGRP-LCx isoform could be due to its ability to bind specific ligands or because its expression is limited to specific cell types by regulated RNA splicing. Overexpression of either of the PGRP-LC cDNAs rescued inducible expression of the Diptericin-lacZ reporter gene in homozygous ird7¹ mutant animals, confirming that the phenotype of ird7 mutants was the result of the lack of PGRP-LC activity (Choe, 2002).

RNA interference (RNAi) was used to test the role of PGRP-LC in the response to bacterial components. Treatment of blood cells from the mbn-2 line with peptidoglycan, Escherichia coli, or lipopolysaccharide (LPS) led to a robust induction of the antibacterial peptide genes. Introduction of double-stranded RNA (dsRNA) of PGRP-LC, but not PGRP-LA, effectively blocked induction of Diptericin, CecropinA1, andAttacinA in response to all three stimuli. Thus, PGRP-LC is required for the response to both peptidoglycan and LPS in these cells (Choe, 2002).

Because PGRP-LC is predicted to encode a transmembrane protein with an extracellular PGRP domain, PGRP-LC may act as a pattern recognition receptor that links recognition of microbial components with host immune responses. Because PGRP-LC is required for responses to both peptidoglycan and LPS, the extracellular domain of PGRP-LC may bind both peptidoglycan and LPS, and binding of either ligand may activate downstream signaling events. Alternatively, PGRP-LC may bind peptidoglycan (but not LPS) and may act as an essential subunit of a larger complex that includes other pattern recognition receptors that bind LPS. In mammals, signaling by Toll-like receptor 2 (TLR2) is activated by peptidoglycan. PGRP-LC might act in a complex with another transmembrane protein similar to TLR2 (Choe, 2002).

Twelve PGRP genes have been identified in the Drosophila genome. Another Drosophila gene, PGRP-SA, encodes a soluble peptidoglycan recognition protein that is essential for activation of the Toll signaling pathway in response to infection by Gram-positive bacteria. Four PGRP genes have already been identified in the human genome. Given the evolutionary conservation of many proteins required for innate immune responses, it will be important to evaluate whether PGRPs function as a family of pattern recognition receptors in human innate immune responses (Choe, 2002).

Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli

The recognition and phagocytosis of microbes by macrophages is a principal aspect of innate immunity that is conserved from insects to humans. Drosophila melanogaster has circulating macrophages that phagocytose microbes similarly to mammalian macrophages, suggesting that insect macrophages can be used as a model to study cell-mediated innate immunity. A double-stranded RNA interference-based screen was devised in macrophage-like Drosophila S2 cells, and 34 gene products involved in phagocytosis have been defined. These include proteins that participate in haemocyte development, vesicle transport, actin cytoskeleton regulation and a cell surface receptor. This receptor, Peptidoglycan recognition protein LC (PGRP-LC), is involved in phagocytosis of Gram-negative but not Gram-positive bacteria. Drosophila humoral immunity also distinguishes between Gram-negative and Gram-positive bacteria through the Imd and Toll pathways, respectively; however, a receptor for the Imd pathway has not been identified. This study shows that PGRP-LC is important for antibacterial peptide synthesis induced by Escherichia coli both in vitro and in vivo. Furthermore, totem mutants, which fail to express PGRP-LC, are susceptible to Gram-negative (E. coli), but not Gram-positive, bacterial infection. These results demonstrate that PGRP-LC is an essential component for recognition and signalling of Gram-negative bacteria. Furthermore, this functional genomic approach is likely to have applications beyond phagocytosis (Ramet, 2002).

The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein>

The antimicrobial defence of Drosophila relies largely on the challenge-induced synthesis of an array of potent antimicrobial peptides by the fat body. The defence against Gram-positive bacteria and natural fungal infections is mediated by the Toll signalling pathway, whereas defense against Gram-negative bacteria is dependent on the Immune deficiency (IMD) pathway. Loss-of-function mutations in either pathway reduce the resistance to corresponding infections. The link between microbial infections and activation of these two pathways has remained elusive. The Toll pathway is activated by Gram-positive bacteria through a circulating Peptidoglycan recognition protein (PGRP-SA). PGRPs appear to be highly conserved from insects to mammals, and the Drosophila genome contains 13 members. This study reports a mutation in a gene coding for a putative transmembrane protein, PGRP-LC, which reduces survival to Gram-negative sepsis but has no effect on the response to Gram-positive bacteria or natural fungal infections. By genetic epistasis, it was demonstrated that PGRP-LC acts upstream of the imd gene. The data on PGRP-SA with respect to the response to Gram-positive infections, together with the present report, indicate that the PGRP family has a principal role in sensing microbial infections in Drosophila (Gotter, 2002).

A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster

Peptidoglycans from bacterial cell walls trigger immune responses in insects and mammals. A peptidoglycan recognition protein, PGRP, has been cloned from moths as well as vertebrates and has been shown to participate in peptidoglycan-mediated activation of prophenoloxidase in the silk moth. This study reports that Drosophila expresses 12 PGRP genes, distributed in 8 chromosomal loci on the 3 major chromosomes. By analyzing cDNA clones and genomic databases, these were grouped into two classes: PGRP-SA, SB1, SB2, SC1A, SC1B, SC2, and SD, with short transcripts and short 5'-untranslated regions; and PGRP-LA, LB, LC, LD, and LE, with long transcripts and long 5'-untranslated regions. The predicted structures indicate that the first group encodes extracellular proteins and the second group, intracellular and membrane-spanning proteins. Most PGRP genes are expressed in all postembryonic stages. Peptidoglycan injections strongly induce five of the genes. Transcripts from the different PGRP genes were found in immune competent organs such as fat body, gut, and hemocytes. It was demonstrated that at least PGRP-SA and SC1B can bind peptidoglycan, and a function in immunity is likely for this family (Werner, 2000).

Search PubMed for articles about Drosophila PGRP-LC

Aggarwal, K., Rus, F., Vriesema-Magnuson, C., Erturk-Hasdemir, D., Paquette, N. and Silverman, N. (2008). Rudra interrupts receptor signaling complexes to negatively regulate the IMD pathway. PLoS Pathog 4: e1000120. PubMed ID: 18688280

Akhouayri, I., Turc, C., Royet, J. and Charroux, B. (2011). Toll-8/Tollo negatively regulates antimicrobial response in the Drosophila respiratory epithelium. PLoS Pathog. 7(10): e1002319. PubMed ID: 22022271

Basbous, N., Coste, F., Leone, P., Vincentelli, R., Royet, J., Kellenberger, C. and Roussel, A. (2011). The Drosophila peptidoglycan-recognition protein LF interacts with peptidoglycan-recognition protein LC to downregulate the Imd pathway. EMBO Rep 12: 327-333. PubMed ID: 21372849

Carty, M., Goodbody, R., Schroder, M., Stack, J., Moynagh, P. N. and Bowie, A. G. (2006). The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling. Nat Immunol 7: 1074-1081. PubMed ID: 16964262

Chang, C. I., Ihara, K., Chelliah, Y., Mengin-Lecreulx, D., Wakatsuki, S. and Deisenhofer, J. (2005). Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition. Proc Natl Acad Sci U S A 102: 10279-10284. PubMed ID: 16006509

Chang, C. I., Chelliah, Y., Borek, D., Mengin-Lecreulx, D. and Deisenhofer, J. (2006). Structure of tracheal cytotoxin in complex with a heterodimeric pattern-recognition receptor. Science 311: 1761-1764. PubMed ID: 16556841

Choe, K. M., Werner, T., Stoven, S., Hultmark, D. and Anderson, K. V. (2002). Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science 296: 359-362. PubMed ID: 11872802

Choe, K. M., Lee, H. and Anderson, K. V. (2005). Drosophila peptidoglycan recognition protein LC (PGRP-LC) acts as a signal-transducing innate immune receptor. Proc. Natl. Acad. Sci. 102: 1122-1126. PubMed ID: 15657141

Dziarski, R. (2004). Peptidoglycan recognition proteins (PGRPs). Mol. Immunol. 40: 877-886. PubMed ID: 14698226

Garver, L. S., Wu, J. and Wu, L. P. (2006). The peptidoglycan recognition protein PGRP-SC1a is essential for Toll signaling and phagocytosis of Staphylococcus aureus in Drosophila. Proc. Natl. Acad. Sci. 103(3): 660-5. PubMed ID: 16407137

Gottar, M., Gobert, V., Michel, T., Belvin, M., Duyk, G., Hoffmann, J. A., Ferrandon, D. and Royet, J. (2002). The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature 416: 640-644. PubMed ID: 11912488

Guan, R., Roychowdhury, A., Ember, B., Kumar, S., Boons, G. J. and Mariuzza, R. A. (2004). Structural basis for peptidoglycan binding by peptidoglycan recognition proteins. Proc Natl Acad Sci U S A 101: 17168-17173. PubMed ID: 15572450

Harris, N., Braiser, D. J., Dickman, D. K., Fetter, R. D., Tong, A. and Davis, G. W. (2015). The innate immune receptor PGRP-LC controls presynaptic homeostatic plasticity. Neuron 88: 1157-1164. PubMed ID: 26687223

Kaneko, T., Goldman, W. E., Mellroth, P., Steiner, H., Fukase, K., Kusumoto, S., Harley, W., Fox, A., Golenbock, D. and Silverman, N. (2004). Monomeric and polymeric gram-negative peptidoglycan but not purified LPS stimulate the Drosophila IMD pathway. Immunity 20: 637-649. PubMed ID: 15142531

Kaneko, T., Yano, T., Aggarwal, K., Lim, J. H., Ueda, K., Oshima, Y., Peach, C., Erturk-Hasdemir, D., Goldman, W. E., Oh, B. H., Kurata, S. and Silverman, N. (2006). PGRP-LC and PGRP-LE have essential yet distinct functions in the drosophila immune response to monomeric DAP-type peptidoglycan. Nat Immunol 7: 715-723. PubMed ID: 16767093

Kim, M. S., Byun, M. and Oh, B. H. (2003). Crystal structure of peptidoglycan recognition protein LB from Drosophila melanogaster. Nat Immunol 4: 787-793. PubMed ID: 12845326

Kleino, A., Myllymaki, H., Kallio, J., Vanha-aho, L. M., Oksanen, K., Ulvila, J., Hultmark, D., Valanne, S. and Ramet, M. (2008). Pirk is a negative regulator of the Drosophila Imd pathway. J Immunol 180: 5413-5422. PubMed ID: 18390723

Kurz, C. L., Charroux, B., Chaduli, D., Viallat-Lieutaud, A. and Royet, J. (2017). Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition. Elife 6. PubMed ID: 28264763

Leulier, F., Vidal, S., Saigo, K., Ueda, R. and Lemaitre, B. (2002). Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults. Curr Biol 12: 996-1000. PubMed ID: 12123572

Leulier, F., Parquet, C., Pili-Floury, S., Ryu, J. H., Caroff, M., Lee, W. J., Mengin-Lecreulx, D. and Lemaitre, B. (2003). The Drosophila immune system detects bacteria through specific peptidoglycan recognition. Nat Immunol 4: 478-484. PubMed ID: 12692550

Lhocine, N., Ribeiro, P. S., Buchon, N., Wepf, A., Wilson, R., Tenev, T., Lemaitre, B., Gstaiger, M., Meier, P. and Leulier, F. (2008). PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling. Cell Host Microbe 4: 147-158. PubMed ID: 18692774

Maillet, F., Bischoff, V., Vignal, C., Hoffmann, J. and Royet, J. (2008). The Drosophila peptidoglycan recognition protein PGRP-LF blocks PGRP-LC and IMD/JNK pathway activation. Cell Host Microbe 3: 293-303. PubMed ID: 18474356

Meinander, A., et al. (2012). Ubiquitylation of the initiator caspase DREDD is required for innate immune signalling. EMBO J. 31(12): 2770-83. PubMed ID: 22549468

Meister, S., Agianian, B., Turlure, F., Relogio, A., Morlais, I., Kafatos, F. C. and Christophides, G. K. (2009). Anopheles gambiae PGRPLC-mediated defense against bacteria modulates infections with malaria parasites. PLoS Pathog 5: e1000542. PubMed ID: 19662170

Mellroth, P., Karlsson, J. and Steiner, H. (2003). A scavenger function for a Drosophila peptidoglycan recognition protein. J. Biol. Chem. 278: 7059-7064. PubMed ID: 12496260

Mellroth, P., Karlsson, J., Hakansson, J., Schultz, N., Goldman, W. E. and Steiner, H. (2005). Ligand-induced dimerization of Drosophila peptidoglycan recognition proteins in vitro. Proc Natl Acad Sci U S A 102: 6455-6460. PubMed ID: 15843462

Neyen, C., Poidevin, M., Roussel, A. and Lemaitre, B. (2012). Tissue- and ligand-specific sensing of gram-negative infection in drosophila by PGRP-LC isoforms and PGRP-LE. J Immunol. 189(4): 1886-97. PubMed ID: 22772451

Neyen, C., Runchel, C., Schupfer, F., Meier, P. and Lemaitre, B. (2016). The regulatory isoform rPGRP-LC induces immune resolution via endosomal degradation of receptors. Nat Immunol 17(10):1150-8. PubMed ID: 27548432

Ramet, M., Manfruelli, P., Pearson, A., Mathey-Prevot, B. and Ezekowitz, R. A. (2002). Functional genomic analysis of phagocytosis and identification of a Drosophila receptor for E. coli. Nature 416: 644-648. PubMed ID: 11912489

Schmidt, R. L., Trejo, T. R., Plummer, T. B., Platt, J. L. and Tang, A. H. (2008). Infection-induced proteolysis of PGRP-LC controls the IMD activation and melanization cascades in Drosophila. FASEB J 22: 918-929. PubMed ID: 18308747

Schmidt, R. L., Rinaldo, F. M., Hesse, S. E., Hamada, M., Ortiz, Z., Beleford, D. T., Page-McCaw, A., Platt, J. L. and Tang, A. H. (2011). Cleavage of PGRP-LC receptor in the Drosophila IMD pathway in response to live bacterial infection in S2 cells. Self Nonself 2: 125-141. PubMed ID: 22496930

Steiner, H. (2004). Peptidoglycan recognition proteins: on and off switches for innate immunity. Immunol. Rev. 198: 83-96. PubMed ID: 15199956

Stensmyr, M. C., Dweck, H. K., Farhan, A., Ibba, I., Strutz, A., Mukunda, L., Linz, J., Grabe, V., Steck, K., Lavista-Llanos, S., Wicher, D., Sachse, S., Knaden, M., Becher, P. G., Seki, Y. and Hansson, B. S. (2012). A conserved dedicated olfactory circuit for detecting harmful microbes in Drosophila. Cell 151(6): 1345-1357. PubMed ID: 23217715

Takehana, A., Yano, T., Mita, S., Kotani, A., Oshima, Y. and Kurata, S. (2004). Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J 23: 4690-4700. PubMed ID: 15538387

Tanji, T., Hu, X., Weber, A. N. and Ip, Y. T. (2007). Toll and IMD pathways synergistically activate an innate immune response in Drosophila melanogaster. Mol Cell Biol 27: 4578-4588. PubMed ID: 17438142

Vaz, F., Kounatidis, I., Covas, G., Parton, R. M., Harkiolaki, M., Davis, I., Filipe, S. R. and Ligoxygakis, P. (2019). Accessibility to peptidoglycan is important for the recognition of gram-positive bacteria in Drosophila. Cell Rep 27(8): 2480-2492. PubMed ID: 31116990

Werner, T., Liu, G., Kang, D., Ekengren, S., Steiner, H. and Hultmark, D. (2000). A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster. Proc Natl Acad Sci U S A 97: 13772-13777. PubMed ID: 11106397

Werner, T., Borge-Renberg, K., Mellroth, P., Steiner, H. and Hultmark, D. (2003). Functional diversity of the Drosophila PGRP-LC gene cluster in the response to lipopolysaccharide and peptidoglycan. J. Biol. Chem. 278: 26319-26322. PubMed ID: 12777387

Zaidman-Remy, A., Herve, M., Poidevin, M., Pili-Floury, S., Kim, M. S., Blanot, D., Oh, B. H., Ueda, R., Mengin-Lecreulx, D. and Lemaitre, B. (2006). The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24: 463-473. PubMed ID: 16618604

date revised: 15 August 2023

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