InteractiveFly: GeneBrief

Peptidoglycan recognition protein SA: Biological Overview | References

The gut sets the immune and metabolic parameters for the survival of commensal bacteria. This study reports that in Drosophila, deficiency in bacterial recognition upstream of Toll/NF-κB signalling resulted in reduced density and diversity of gut bacteria. Translational regulation factor 4E-BP (Thor), a transcriptional target of Toll/NF-κB, mediated this host-bacteriome interaction. In healthy flies, Toll activated 4E-BP, which enabled fat catabolism, which resulted in sustaining of the bacteriome. The presence of gut bacteria kept Toll signalling activity thus ensuring the feedback loop of their own preservation. When Toll activity was absent, TOR-mediated suppression of 4E-BP made fat resources inaccessible and this correlated with loss of intestinal bacterial density. This could be overcome by genetic or pharmacological inhibition of TOR, which restored bacterial density. These results give insights into how an animal integrates immune sensing and metabolism to maintain indigenous bacteria in a healthy gut (Bahuguna, 2022).

The animal gut accommodates a diverse array of bacteria, which assist in regulation of digestion, supply of nutrients and metabolites as well as in immune development. To reap benefits from these microbes, the host provides a symbiotic environment for sustaining them in the gut. The Drosophila gut and its bacteriome is used as a simpler model to study such host-microbe interactions. Although much less diverse compared to humans, the fly bacteriome is equally dynamic and changes with age and environmental conditions connected to reinfections during fly culture (Bahuguna, 2022).

The Drosophila intestinal epithelium is immunocompetent and upon enteric infection initiates innate immune responses via the NF-κB pathway IMD, mediating the production of antimicrobial peptides (AMPs) as well as the pathway centered on Dual Oxidase, the enzyme needed for the generation of Reactive Oxygen Species (ROS). However, it also preserves commensal bacteria, since transcription of AMPs is suppressed by Caudal and Nubbin, while bacterial-derived uracil is important for distinguishing pathogens from commensals (Bahuguna, 2022).

The evolutionary conserved Target of Rapamycin (TOR) pathway is a major pathway controlling cellular metabolism and growth. TOR balances lipid and glucose anabolism and catabolism in the cell through the activity of the TORC1 protein complex. TORC1 promotes protein synthesis primarily through phosphorylation of the eIF4E Binding Protein (4E-BP/Thor) and p70S6 Kinase 1 (S6K1). 4E-BP is a translational inhibitor, which binds and inhibits the activity of eIF4E an eukaryotic translation initiation factor responsible for the recruitment of 40s ribosomal subunit at the 5'-cap of mRNA. Phosphorylation of 4E-BP lowers its affinity towards eIF4E. This frees eIF4E, enabling it to promote cap-dependent translation. In the case of S6K1, activated S6K1 promotes protein synthesis by activating inducers of mRNA translation initiation whilst degrading inhibitors (Bahuguna, 2022).

Drosophila TOR has been extensively studied for its role in growth and development, using fly mutants or by treating flies with rapamycin. Rapamycin treatment in stress conditions, led to upregulation of 4E-BP activity resulting in an increase of whole-fly lipid reserves that could be used for the long-term survival of these stress conditions. In contrast, 4E-BP mutants were unable to preserve lipid stores and had thus compromised survival following starvation or oxidative stress. More broadly, the consensus is that in both Drosophila and mice, 4E-BP regulates fat levels in stress conditions like starvation and oxidative stress. During larval stages and when in food with poor nutritional value, the presence of the commensal Lactobacillus plantarum is important to sustain development through TOR, which is in turn crucial for sustaining this mutualistic relationship. The metabolic state of the gut is also influenced by dietary conditions and in its turn influences the bacteriome. Diet-dependent adaptations of the microbiota require NF-κB-dependent control of the translational regulator 4E-BP and this where TOR and NF-κB 'meet' (Bahuguna, 2022).

Drosophila has three NF-κB proteins namely, Relish, Dorsal and the Dorsal-related Immunity Factor (DIF). DIF is downstream of the Toll signalling pathway. Toll and Toll-like receptor (TLR) signalling is one of the most important evolutionary conserved mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms. Unlike its mammalian counterparts however, Drosophila Toll is activated by an endogenous cytokine-like ligand, the Nerve Growth Factor homologue, Spz. Spz is processed to its active form by the Spz-Processing Enzyme (SPE). Two serine protease cascades converge on SPE: one triggered by bacterial or fungal serine proteases through the host serine protease Persephone and a second activated by host receptors that recognise bacterial or fungal cell wall. Prominent among these host receptors is the Peptidoglycan Recognition Protein-SA or PGRP-SA. PGRP-SA binds to peptidoglycan (PG) on the bacterial cell wall without structural preference but depending on accessibility and generates the downstream signal (Bahuguna, 2022).

When the recognition signal reaches the cell surface, it is communicated intracellularly via the Toll receptor and a membrane-bound receptor-adaptor complex including dMyd88, Tube (as an IRAK4 functional equivalent) and the Pelle kinase (as an IRAK1 functional homologue). Transduction of the signal culminates in the phosphorylation of the IκB homologue, Cactus probably by Pelle, leaving the NF-κB homologue DIF to move to the nucleus and regulate hundreds of target genes including antimicrobial peptides (AMPs) (Bahuguna, 2022).

In this study, evidence is presented that PGRP-SA is important for the preservation of commensal intestinal bacterial density. The results reveal that larvae and adults that are deficient in PGRP-SA or DIF have a significantly reduced commensal gut bacterial density. Inhibition of the activity of TOR by Rapamycin or TOR RNAi in enterocytes, restores bacterial density (but not diversity) in PGRP-SA or DIF mutant guts. However, flies mutants for PGRP-SA and deficient for 4EBP in enterocytes were unable to restore bacterial density upon Rapamycin treatment or TOR RNAi, demonstrating the important role of 4EBP. PGRP-SA mutants had increased intestinal fat stores that were restored to normal levels through Rapamycin or TOR-RNAi treatment in enterocytes. This restoration failed in PGRP-SA;4EBP double mutants indicating that 4EBP was crucial in regulating fat stores in the gut. Fat catabolism was important for gut bacterial restoration as flies deficient for PGRP-SA and treated with rapamycin were unable to restore bacterial density if the triglyceride lipase Brummer was knocked down in enterocytes. This mechanism gives an insight into how the host integrates immunity and metabolism to maintain commensal bacteria at the intestinal epithelium (Bahuguna, 2022).

In the absence of the bacterial receptor PGRP-SA from enterocytes, a reduction was observed in intestinal bacterial density. It was restored with the use of rapamycin, which targets TORC1 or by knocking-down TOR in enterocytes. This suggested that loss of the immune receptor PGRP-SA, generated a metabolic environment unfavourable for intestinal bacterial growth. The results indicated that at the centre of this relationship was 4E-BP, which is activated by Toll and suppressed by TORC1. In keeping with this, PGRP-SA^seml; NP1GAL4>4E-BPRNAi flies treated with rapamycin were unable to restore gut bacterial density. Intestinal lipid catabolism downstream of 4EBP was paramount for the maintenance of cultivable bacterial density because the loss of the lipase Bmm blocked restoration of gut bacteria after rapamycin treatment. Silencing of bmm in enterocytes caused intestinal lipid accumulation and prevented any restoration via rapamycin in PGRP-SA^seml flies (Bahuguna, 2022).

These results indicate that downstream of Toll, intestinal triglyceride levels were under 4E-BP control in enterocytes. Although the phenomenon of cultivable bacteriome reduction in PGRP-SA^seml flies was readily manifested in larvae and young flies, the results indicated that it was also there in older flies. Conventional fly rearing techniques ensure a steady stream of defaecation and re-introduction of bacteria over time. However, when food vials were changed rapidly re-infection was reduced and CFUs in 30-day old flies were significantly lower in PGRP-SA^seml than yw flies. Preservation of the bacteriome was dependent on PG recognition as the rescue of enteric CFUs in PGRP-SA^seml flies was only possible with PGRP-SA transgenes that had an intact PG binding ability. This indicated that bacterial sensing was the initial trigger point to activate the process (Bahuguna, 2022).

A working model is depicted A schematic model outlining the role of PGRP-SA/Toll/Dif in the retention of the gut bacteriome. PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. In turn, this keeps increased 4E-BP transcription/4E-BP protein phosphorylation in enterocytes, preserving a steady rate of intestinal lipid catabolism. The latter is important for maintaining normal density of commensal bacteria. It is hypothesised that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria. In keeping with this, stopping lipid catabolism by silencing the Bmm lipase in ECs resulted in accumulation of lipids and reduction of enteric CFUs. According to the model, bacteria should trigger lipid catabolism and 5-day old axenic flies showed a clear trend for lipid accumulation in their gut, but this was marginally not statistically significant. Studies with Vibrio cholera, have shown that intestinal acetate leads to deactivation of host insulin signalling and lipid accumulation in enterocytes, resulting in host lethality. Loss of PGRP-SA/Dif leads to a decrease in lifespan. Whether this is due to the long-term accumulation of lipids is an open question (Bahuguna, 2022).

PGRP-SA recognises components of the intestinal bacteriome and activates the Toll pathway in enterocytes. This increases 4E-BP transcription/4E-BP protein phosphorylation in enterocytes. 4EBP is important for maintaining normal density of commensal bacteria. It is hypothesized that Bmm-mediated lipid catabolism is regulated by 4E-BP and released triglycerides act as fuel for the maintenance of commensal bacteria (Bahuguna, 2022).

More work is needed to understand whether/how stored intestinal lipids maybe released to circulation, how commensal bacteria receive them and which component(s) of the bacteriome are recognised by PGRP-SA (Bahuguna, 2022).

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).

A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway

The Drosophila Toll receptor does not interact directly with microbial determinants, but is instead activated by a cleaved form of the cytokine-like molecule Spätzle. During the immune response, Spätzle is processed by complex cascades of serine proteases, which are activated by secreted pattern-recognition receptors. This study demonstrates the essential role of ModSP, a modular serine protease, in the activation of the Toll pathway by gram-positive bacteria and fungi. ModSP integrates signals originating from the circulating recognition molecules GNBP3 and PGRP-SA and connects them to the Grass-SPE-Spätzle extracellular pathway upstream of the Toll receptor. It also reveals the conserved role of modular serine proteases in the activation of insect immune reactions (Buchon, 2009).

Sequential activation of extracellular serine protease (SP) cascades regulates important innate immune reactions, like blood clotting in arthropods and complement activation in vertebrates. In Drosophila, proteolytic cascades are also involved in the regulation of the Toll pathway, which mediates resistance to Gram-positive bacteria and fungi. Unlike mammalian Toll-like receptors, the Drosophila Toll receptor does not interact directly with microbial determinants and is instead activated by a cleaved form of the secreted cytokine-like molecule Spätzle (Spz). The immune-induced cleavage of Spz is triggered by proteolytic cascades, conceptually similar to vertebrate blood coagulation or complement activation cascades. These proteolytic cascades have a functional core consisting of several SP that undergo zymogen activation, upon cleavage by an upstream protease. Spätzle processing enzyme (SPE), an immune-regulated SP with a Clip-domain, has been identified as the terminal SP that maturates Spz (Jang, 2006; Kambris, 2006 and Bouchon, 2009 and references therein).

Genetic analysis supports the existence of several complex cascades of SPs that link microbial recognition to activation of SPE. Pattern-recognition receptors (PRRs) are thought to be present in the hemolymph where they sense microbial-derived molecules. Detection of Gram-positive bacteria is mediated through the recognition of peptidoglycan by the peptidoglycan-recognition protein-SA (PGRP-SA) with the help of Gram-negative binding protein1 (GNBP1). Activation of the Toll pathway by fungi is in part mediated by GNBP3 through the sensing of β-1,3-glucan (Gottar, 2006). RNAi and loss of function analyses have shown that activation of SPE by either PGRP-SA/GNBP1 or GNBP3 requires Grass, a Clip-domain SP ( Bouchon, 2009 and references therein).

Spores of entomopathogenic fungi such as Beauveria bassiana have the capacity to germinate on the fly cuticle and generate hyphae, which can penetrate the cuticle of insects and reach the hemolymph. It has been proposed that the presence of B. bassiana is detected, in a GNBP3-independent manner, through direct activation of the Toll pathway by a fungal protease PR1. PR1 would directly cleave the host SP, Persephone (Psh), which triggers Toll pathway activation. Recently, this mode of activation was extended to the sensing of proteases produced by various bacteria. Surprisingly, tracheal melanization in mutant larvae lacking the serpin Spn77Ba also activates the Toll pathway in a Psh-dependent manner (Tang, 2008). This suggests that Psh-dependent Toll pathway activation is induced by a host factor derived from melanization. This also points to a possible cross-talk between the proteolytic cascades that regulate the Toll pathway and those regulating the melanization reaction (Bouchon, 2009).

Despite these recent studies, the extracellular events that lead to the activation of the Toll pathway remain poorly characterized. For instance, the apical protease linking recognition by PRRs to the cleavage of Spz is not known, leaving an important gap in knowledge of Toll pathway activation. The in vitro reconstruction of a similar cascade with purified proteases in the beetle Tenebrio molitor, suggests an important role for a modular SP (Tm-MSP). In this insect, binding of peptidoglycan to the PGRP-SA/GNBP1 complex induces activation of the Tm-MSP zymogen that in turn activates another SP (Tm-SAE) to cleave Tm-SPE, the Tenebrio homolog of SPE, resulting in both activation of Spz and melanization (Bouchon, 2009 and references therein).

This result prompted an investigation of the role of the Drosophila homolog of Tm-MSP, which is encoded by the CG31217 gene. A null mutation was generated in CG31217 by homologous recombination and its essential role in the activation of Toll by secreted PRRs was demonstrated (Bouchon, 2009).

modSP deficient flies fail to activate the Toll pathway in response to either Gram-positive bacteria or yeast. Epistatic analyses demonstrated that ModSP acts downstream of PGRP-SA, but upstream of the SP Grass. Importantly, ModSP was shown not to participate in the Psh-dependent branch of the Toll pathway as shown by the wild-type activation of this pathway in modSP¹ flies upon injection of bacterial proteases. The modSP¹ phenotype was very similar to that of a loss of function Grass mutant, suggesting that these SP act in a linear pathway connecting microbe recognition by PRR to the activation of Spz by SPE. The analysis of psh¹; modSP¹ double mutant flies indicates a synergistic action of the ModSP and Psh pathways in the response against filamentous fungi, which might be detected through both host PRRs and their virulence factors. Collectively, these results confirm the model of an activation of Toll by 2 extracellular pathways: A PRR-dependent pathway and a Psh-dependent pathway. This model was extended by showing that Grass and ModSP function in a common SP cascade. In addition, the apical position of ModSP suggests a direct branching of signals from secreted PRRs to the ModSP-Grass pathway. Biochemical analyses in T. molitor indicate that Tm-MSP interacts directly with the PRR complexes involved in the sensing of peptidoglycan or glucan. Although it was not formally demonstrated in the present study, it seems likely that Drosophila ModSP acts as the most upstream SP directly activated by secreted PRRs (Bouchon, 2009).

ModSP has a number of structural features that make it unique among Drosophila SP and suggest its critical role in the initial events leading to the activation of the proteolytic cascade upstream of Toll. First, ModSP does not contain a Clip-domain in contrast to Grass and SPE. The function of the Clip-domain is still unknown but its presence in many SP that function in signaling cascades suggests a regulatory role (Piao, 2005). This indicates that ModSP activation differs from the chain reaction activating Grass or SPE. Second, ModSP contains additional domains such as the CCP and LDLa motifs in its N-terminal extremity. Consistent with the presence of the LDLa domain, a ModSP-GFP fusion protein was secreted and found bound to the surface of lipid vesicles that circulate in the hemolymph. It is speculated that the association of ModSP to vesicles is important to nucleate the activation of downstream SP (Bouchon, 2009).

In the beetle, T. molitor, Tm-MSP is activated upon formation of a tri-molecular complex composed of PGRP-SA, GNBP1, and lysine-type peptidoglycan (Kim, 2008). Although the possibility of a SP other than ModSP functioning as the initial protease in the Drosophila Toll pathway cannot be ruled out, epistatic analysis suggests an apical role of ModSP. Overexpression of a full-length version of ModSP is sufficient to reach a high level of Toll activation, in contrast to other SP that generally require the overexpression of a preactivated form to fully induce the cascade. These observations favor a model in which ModSP directly interacts with GNBP3 or GNBP1/PGRP-SA recognition complexes. Recruitment of ModSP by PRRs would increase its local concentration, a situation sufficient for its autoproteolysis. In agreement with this hypothesis, a recombinant form of ModSP produced in baculovirus appears to be unstable as a zymogen, presumably because of a high level of autoactivation. Unfortunately, this high level of autoproteolysis did not permit in vitro reconstitution experiments using ModSP, GNBP1, and PGRP-SA as performed with Tm-MSP. Nevertheless, a higher level of ModSP activation was observed when the protein was incubated with GNBP1 and PGRP-SA. The most parsimonious model is that ModSP would interact with GNBP1 or GNBP3, the common protein family members found in the 2 recognition complexes. The exact contribution of GNBP1 to sensing Gram-positive bacteria is currently debated. It was recently proposed that GNBP1 functions upstream of PGRP-SA by cleaving peptidoglycan, a step required for an optimal binding of PGRP-SA to peptidoglycan. In contrast, T. molitor GNBP1 is recruited subsequent to the binding of PGRP-SA to peptidoglycan and is required for the interaction with Tm-MSP. The implication of ModSP in sensing of fungi through GNBP3 and peptidoglycan through GNBP1/PGRP-SA suggests a similar mechanism of activation of this SP and Tm-MSP. This would favor a role of GNBP1 as a linker between PGRP-SA and ModSP. In accordance with this model, neither recombinant full-length Drosophila nor Tenebrio GNBP1 exhibited any enzymatic activity toward peptidoglycan in vitro (Bouchon, 2009).

The participation of ModSP and SPE in an extracellular pathway linking PRR recognition to Spz activation in both T. molitor (Coleoptera) and D. melanogaster (Diptera), which diverged ~250 million years ago, demonstrates the conservation of this extracellular signaling module in insects. In the lepidopteran Manduca sexta, the hemolymph protein 14 (Ms-HP14) contains a domain arrangement very similar to that of ModSP and regulates the melanization cascade in response to microbial infection. Collectively, biochemical studies performed in T. molitor and M. sexta, and genetic analysis in D. melanogaster, reveal striking similarities in the mechanisms underlying SP activation by PRRs. All involve the sequential activation of an apical modular SP (that displays a certain level of autoactivation) and clip-domain proteases. Interestingly, a similar organization is also observed in the proteolytic cascade that regulates Toll during dorso-ventral patterning of the embryo. Gastrulation Defective, the apical SP, contains von Willebrand domains and is also thought to be autoactivated, although the precise mechanism that triggers its activation has not been determined. These features are also reminiscent of the complement activation by the lectin pathway in mammals in which the recognition of carbohydrate by the mannose binding lectin (MBL) leads to the autoactivation of MBL-associated serine proteases (MASPs). MASPs are also modular proteases with CUB, CCP, and EGF domains in their N terminus. Thus, genetic and biochemical analyses now reveal a similar level of organization for various proteolytic cascades in insects and increase understanding as to how these cascades have evolved to fulfill diverse developmental or immune functions (Bouchon, 2009 and references therein).

A complete understanding of the precise sequence of events leading to Toll pathway activation is still in the future, because the task is complicated by the high number of SP encoded in the Drosophila genome. The analysis described in this paper strongly suggests the existence of an intermediate SP acting between ModSP and Grass. Future works should identify this SP and determine the interaction between Grass and SPE. To date, no serpin regulating SP involved in the PRR-dependent pathways has been identified despite the critical role of this family in the negative control of proteolytic cascades. Further experiments combining genetics, biochemistry, and cell biology are required to identify additional components of this cascade and to clarify in vivo how and where proteolytic cascades downstream of PGRP-SA or GNBP3 are activated in the hemolymph compartment (Bouchon, 2009).

Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors

The Drosophila immune system discriminates between various types of infections and activates appropriate signal transduction pathways to combat the invading microorganisms. The Toll pathway is required for the host response against fungal and most Gram-positive bacterial infections. The sensing of Gram-positive bacteria is mediated by the Pattern Recognition Receptors PGRP-SA and Gram-negative bacteria binding protein 1 (GNBP1) that cooperate to detect the presence of infections in the host. This study report that Gram-negative bacteria binding protein 3 (GNBP3) is a novel Pattern Recognition Receptor that is required for the detection of fungal cell wall components. Strikingly, there is a second, parallel, pathway acting jointly with GNBP3. The Drosophila Persephone protease activates the Toll pathway when proteolytically matured by the secreted fungal virulence factor PR1. Thus, the detection of fungal infections in Drosophila relies both on the recognition of invariant microbial patterns and on monitoring the effects of virulence factors on the host (Gottar, 2006).

Fungi represent a threat to insects in the wild with more than 700 entomopathogenic species described. Insects must have evolved responses to handle these infections. This study attempts to decipher the mechanisms that stimulate immune responses of Drosophila to fungal infections (Gottar, 2006).

This host response includes both cellular and humoral arms. The analysis of the humoral immune response within the framework of a septic injury model has led to the current paradigm in which two distinct intracellular transduction pathways, Immune deficiency (IMD) and Toll, regulate the transcription of hundreds of genes by controlling the nuclear uptake of the NF-κB transcription factors Relish and Dorsal-related Immunity Factor (DIF), respectively. The classical effector molecules of the systemic humoral response, the antimicrobial peptides, are synthesized in the fat body, a functional analogue of the mammalian liver, and are released into the hemolymph where they kill invading microorganisms. One of these peptides, Drosomycin, exhibits fungicidal activities at micromolar concentrations and is active mainly on filamentous fungi. Others such as Cecropins, Attacins, Drosocin, and Diptericin are active mostly on Gram-negative bacteria whereas Defensin is effective against Gram-positive bacteria (Gottar, 2006).

The IMD pathway is required for the host response against Gram-negative bacteria. Mutants in this pathway fail to express antibacterial peptides and are highly sensitive to such infections, yet resist fungal and Gram-positive bacterial infections as well as wild-type flies. Toll is the receptor of the second intracellular transduction pathway and is activated by the binding of a proteolytically cleaved form of the Spätzle (SPZ) cytokine. Toll pathway mutants are susceptible to infections by the entomopathogenic fungi B. bassiana and M. anisopliae or by the opportunistic pathogen Aspergillus fumigatus. Toll is also required to resist some Gram-positive bacterial infections (Gottar, 2006).

The Drosophila immune response is adapted to the nature of the invading microorganism. The Toll pathway is induced by fungi and Gram-positive bacteria, whereas the IMD pathway is predominantly triggered upon Gram-negative bacterial challenges. These observations imply that several receptors mediate the discrimination between various types of microbial infections. Indeed, members of the peptidoglycan recognition protein (PGRP) family have been shown to be required for these distinct events (reviewed in Ferrandon, 2004; Kaneko, 2005). PGRP-LC, a receptor of the IMD pathway, can be activated by meso-diaminopimelic acid peptidoglycan (PGN), a compound characteristic of the cell wall of Gram-negative bacteria and of Gram-positive bacilli. Peptidoglycan recognition protein LE (PGRP-LE), a secreted member of the PGRP family, is also involved in sensing Gram-negative bacteria (Kaneko, 2006). In contrast, the circulating PGRP-SA receptor activates the Toll pathway upon detection of Lysine-type PGN which is a major component of the cell wall of many Gram-positive bacterial strains. The Gram-Negative Binding Protein 1 (GNBP1) associates with PGRP-SA and this complex is both necessary and sufficient to activate the Toll pathway upon Gram-positive challenge (Gobert, 2003). The circulating PGRP-SA/GNBP1 complex activates a downstream proteolytic cascade that leads to the cleavage of the Spätzle cytokine, which then activates the Toll transmembrane receptor (Jang, 2006; Kambris, 2006). Thus, PGRP-SA and GNBP1 define a Gram-positive-specific branch of Toll receptor activation. PGRP-SD also belongs to this branch and is required for the detection of other Gram-positive bacterial strains (Bischoff, 2004; Gottar, 2006 and references therein).

This study addresses the existence of a second branch devoted to the detection of fungal infections, which also activates Toll. Indeed, mutants for the persephone (psh) gene, which encodes a clip-prodomain containing protease, are characterized by an increased sensitivity to natural infections with the entomopathogenic fungus B. bassiana, whereas they are resistant to bacterial infections. The psh mutations had been originally isolated as suppressors of the necrotic (nec) phenotype. nec encodes a serine protease inhibitor of the serpin family, the absence of which leads to the constitutive, psh-dependent, activation of the Toll pathway (Levashina, 1999; Ligoxygakis, 2002). Thus, psh and nec define a fungal-specific branch of Toll receptor activation. By analogy to the Gram-positive branch, it is expected that an as yet unidentified immune receptor detects fungal infections and activates in turn the psh-dependent proteolytic cascade (Gottar, 2006).

GNBP1 belongs to the family of GNBP/β-Glucan Recognition Proteins (βGRP) (Kim, 2000). Members of this family have been reported to bind to β-(1,3)-glucan, a major component of the fungal cell wall (Lee, 2006; Ma, 2000; Ochiai, 2000). In Drosophila, three members of this family, GNBP1 to 3, have been described (Kim, 2000). Among these, GNBP3 shows the greatest degree of similarity to lepidopteran β-(1,3)-glucan recognition proteins and was therefore a good candidate for a fungal-specific sensor. This study reports that GNBP3 is indeed required for Toll pathway activation in response to fungal infections. Strikingly, psh is required in a distinct yet complementary detection pathway that can be activated by fungal virulence factors (Gottar, 2006).

The detection of infections is a crucial step in the timely initiation of an appropriate immune response. In Drosophila, the use of nonentomopathogenic bacteria such as M. luteus and E. coli has allowed the delineation of both intracellular signal transduction pathways as well as the identification of five innate receptors (PRRs), PGRP-LC and -LE for the IMD pathway, and PGRP-SA/GNBP1/PGRP-SD for the Toll pathway. To elucidate the mechanisms involved in the detection of fungi, concentration was placed on a somewhat artificial infection system using an opportunistic human pathogenic yeast, C. albicans. Understanding of the system was refined by using the entomopathogenic fungi B. bassiana and M. anisopliae (Gottar, 2006).

This study demonstrated that GNBP3 is a pattern recognition receptor dedicated to the detection of fungi since (1) recombinant GNBP3 is able to bind in vitro to Candida and to polymeric chains of β-(1,3)-glucan; (2) it is required for the activation of the Toll pathway by polysaccharides of the fungal cell wall; (3) GNBP3 is required for resistance against yeast infections, including C. albicans, C. glabrata, C. tropicalis, and against mold infections such as B. bassiana, M. anisopliae, and A. fumigatus; (4) GNBP3 triggers an adequate immune response, namely it activates the antifungal Toll pathway in a spz-dependent manner. It cannot be formally excluded that another fungal receptor acts together with GNBP3 to activate the Toll antifungal host defense (Gottar, 2006).

Of note is that fungi can induce the IMD pathway with short-term kinetics. This induction is dependent on PGRP-LC and not on GNBP3. One possibility is that a PGRP-LC coreceptor senses fungal microbial patterns. Alternatively, fungal cell wall constituents might bind directly to PGRP-LC. Interestingly, Lee (2003) has reported that a coleopteran PGRP is able, in addition to its liaison to PGN, to bind with high affinity to tetralaminariose, a tetramer of β-(1,3)-glucan (Gottar, 2006).

As is the case for members of the PGRP family, the GNBP/βGRP proteins have evolved to recognize distinct carbohydrate chains that form the cell wall of microorganisms. Given their distribution in the arthropod lineage, it is likely that these two families form an essential part of their immunity repertoire. Whereas PGRP homologs exist in mammals, βGRP members have not been reported in vertebrates. However, the phagocytic and signalling receptor Dectin-1 detects β-(1,3)-glucans (Brown, 2001) and may to some extent fulfill in mammals a primary function that is similar to that of GNBP3 in insects, i. e., the sensing of fungal infections (Gottar, 2006).

Since spz is required for Toll activation by GNBP3, it is proposed that the binding of GNBP3 to its microbial ligand leads to the activation of a proteolytic cascade that ultimately processes proSPZ into a functional Toll ligand. Because psh and GNBP3^hades have distinct phenotypes as regards Toll pathway activation and because the double mutant psh; GNBP3^hades displays a stronger phenotype than either mutant alone when challenged with live fungi, PSH cannot belong exclusively to a proteolytic cascade activated by GNBP3. However, epistatic analysis reveals that the spz-dependent expression of Drosomycin induced by GNBP3 overexpression partly requires psh function. Taken together, these data indicate the existence of an alternative, psh-independent proteolytic cascade that mediates the GNBP3-dependent maturation of the Toll ligand Spätzle. This cascade is distinct from the one that activates Toll signalling during early embryogenesis (Gottar, 2006).

Many pathogens have adapted to their hosts and developed specific strategies to defeat their defenses. Fungi such as B. bassiana and M. anisopliae are able to infect insects following deposition of spores on the surface of the cuticle. To penetrate this physical barrier, they secrete several virulence factors such as chitinases and proteases. The PR1A protease is able to activate Drosomycin expression in the absence of infection when overexpressed in flies. This effect on Toll pathway activation is specific since it can be blocked in a psh background and depends on the proteolytic activity of PR1A. These data establish the proof of concept that a virulence factor can be detected by the innate immune system. Interestingly, these data indicate that PR1 can directly process PSH into its active form (Gottar, 2006).

PR1A is one of ten proteases in this subtilisin family and is expressed only during cuticle penetration. This study found that a PR1A/PR1B-deficient strain is still able to induce Drosomycin expression in a GNBP3^hades mutant background, presumably through other fungal PR1 proteases. Thus, further work will be required to understand the multiple pathogenic mechanisms taking place during a natural fungal infection (Gottar, 2006).

These data show that the detection of fungal infections relies on a two-pronged sensor system that constitutes a partially redundant recognition system. The psh; GNBP3 double mutant strain consistently yields a stronger phenotype than that of the respective single mutants. Since only GNBP3 is strictly required in the defense against opportunistic yeasts, it is likely that the recognition of fungal patterns represents an ancestral, basal mode of infection sensing. The psh-dependent system that monitors virulence factors may have evolved secondarily in response to the selective pressure exerted by entomopathogenic fungi. Indeed, if the psh-based and the GNBP3-based sensing systems were perfectly redundant, it would be expected that the deletion of one of these systems would not prevent the activation of the Toll pathway. This is indeed what was observed when infecting flies with live C. albicans or with M. anisopliae. In contrast, Drosomycin inducibility is abolished in psh mutants, but not in GNBP3 mutants, infected by B. bassiana. These data indicate that B. bassiana has evolved a strategy that allows it to escape or to block GNBP3 surveillance (Gottar, 2006).

Future studies will reveal whether similar systems of virulence factor detection exist also to sense infection by entomopathogenic bacteria (Gottar, 2006).

It is surmised that some pathogens have developed strategies to inactivate the GNBP basal sensor system of Drosophila and that this led to the selection of a novel host counter-strategy : the surveillance of virulence factor activity. This theme is a central tenet of the current understanding of plant innate immunity. In plants, basal sensor systems detect the presence of microbial elicitors and trigger an immune response. Some virulence factors of the plant pathogen inhibit the elicitor-induced signaling by manipulating host proteins that regulate the host basal response. In some plant cultivars, a surveillance system based on R proteins 'guards' the targets of virulence factors (coded by microbial avirulence (avr) genes) and triggers a strong immune response when under attack. One example is provided by Arabidopsis where the cleavage of the endogenous PBS1 kinase by the Pseudomonas syringae type III effector AvrPphB, a cysteine protease, leads to the activation of the hypersensitive response by the R protein RPS5. A case possibly more relevant to fly immunity is provided by the tomato where the host protease Rcr3 is required for the recognition of the pathogen virulence factor Avr2 by the Cf-2 transmembrane receptor (Gottar, 2006).

Fungal proteases secreted by entomopathogenic fungi have to cross the structurally invariant cuticular barrier of the insect host that thus conditions the type of proteolytic activity required to degrade the cuticular proteins. This phenomenon may have been exploited by Drosophila to detect entomopathogenic infections in a mechanism that is hence conceptually related to the guard hypothesis of plants, although in this case, PSH would monitor indirectly a passive defense mechanism, the protection provided by the bodily armor. To date, the analysis of the immune response in Drosophila has been largely limited to the study of laboratory strains in a controlled environment. By analogy to plant-pathogen interactions that involve avr genes and their cognate plant R resistance genes, a major challenge for the coming years will be to determine whether the insect-pathogen interactions in a natural environment involve several distinct virulence factors and their associated host detection systems (Gottar, 2006).

The discovery of a host sensor system dedicated to the detection of virulence factor activity begs the question of the relevance of such a system to mammalian innate immunity. It has been reported that virulence factors such as the cholesterol-dependent cytolysin or pertussis toxin are able to induce immune responses through TLR4. In these cases, the possibility remains open that TLR4 functions as a co-receptor needed for intracellular signal transduction and that the actual recognition is mediated by unknown receptors. A second class of interest is that of the Protease Activated Receptors. Indeed, PAR2 has been implicated in the induction of the HB2 defensin by bacterial proteases in epithelial cells. Similarly, Citrobacter rodentium induces the intestinal release of host proteases that activate the PAR2 receptor and subsequent colonic inflammation. Finally, virulence factors from Salmonella and Yersinia have been shown to inhibit NF-κB and MAPK signaling. Thus, it is legitimate to ask whether receptors dedicated to the perception of virulence factor activity have been selected during the evolution of the mammalian innate immune system (Gottar, 2006 and references therein).

Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein

Microbial infection activates two distinct intracellular signaling cascades in the immune-responsive fat body of Drosophila. Gram-positive bacteria and fungi predominantly induce the Toll signaling pathway, whereas Gram-negative bacteria activate the Imd pathway. Loss-of-function mutants in either pathway reduce the resistance to corresponding infections. Genetic screens have identified a range of genes involved in these intracellular signaling cascades, but how they are activated by microbial infection is largely unknown. Activation of the transmembrane receptor Toll requires a proteolytically cleaved form of an extracellular cytokine-like polypeptide, Spatzle, suggesting that Toll does not itself function as a bona fide recognition receptor of microbial patterns. This is in apparent contrast with the mammalian Toll-like receptors and raises the question of which host molecules actually recognize microbial patterns to activate Toll through Spatzle. A mutation is described in this study that blocks Toll activation by Gram-positive bacteria and significantly decreases resistance to this type of infection. The mutation semmelweis (seml) inactivates the gene encoding a peptidoglycan recognition protein (PGRP-SA). Interestingly, seml does not affect Toll activation by fungal infection, indicating the existence of a distinct recognition system for fungi to activate the Toll pathway (Michel, 2001).

The sequence of the PGRP-SA complementary DNAs from seml and wild-type flies was compared. In the seml cDNA there is a transition of guanine to adenine at position 174, which results in the change of cysteine 80 into a tyrosine. This mutation affects a region of amino acids (called the PGRP domain) that is extremely conserved among the members of the PGRP family of vertebrates and invertebrates. This cysteine is conserved in more than 90% of the PGRPs, a feature shared by only 5% of the amino acids that form the PGRP domain. Furthermore, a cysteine is present in close proximity (Cys 74) that might engage in a disulphide bridge with Cys 80. The seml mutation would disrupt such a bridge and affect the three-dimensional structure of the protein. To ascertain that the mutation of Cys 80 to Tyr 80 is responsible for the seml phenotypes, rescue experiments were undertaken by overexpressing wild-type dPGRP-SA cDNA, using the ubiquitous driver DaughterlessGal4 (DaGal4). Adult seml;DaGal4-UAS PGRP-SA flies have a restored ability to induce drosomycin after M. luteus infection and are no longer susceptible to Gram-positive infection. In contrast, seml;DaGal4 flies show phenotypes identical to seml flies. These results unambiguously demonstrate that the inability of seml mutant flies to resist Gram-positive infection results from the inactivation of the PGRP-SA gene (Michel, 2001).

The deduced sequence of the PGRP-SA gene indicates the presence of a putative signal peptide, suggesting that the PGRP-SA protein is a secreted protein present in the hemolymph or possibly associated with the extracellular matrix. It was reasoned that if the PGRP-SA protein is present in the hemolymph, it should be possible to rescue the seml phenotype by transferring wild-type hemolymph into the mutant flies. Indeed, when wild-type hemolymph was injected into seml flies, the recipient flies became capable of expressing drosomycin after challenge by M. luteus. These results demonstrate that PGRP-SA is a protein circulating in the hemolymph, where the molecular recognition with the Gram-positive bacterial cell wall components can occur (Michel, 2001).

The data demonstrate that activation of the Toll pathway by Gram-positive bacterial infection is mediated by a circulating peptidoglycan recognition protein. This is the first demonstration of an in vivo function for a pattern-recognition receptor in invertebrate innate immunity. In view of the relatively large number of genes (12) encoding peptidoglycan recognition proteins in Drosophila, it was surprising to see that the mutation of one of these genes was sufficient to abolish Toll activation by Gram-positive bacteria. No other circulating protein can substitute for PGRP-SA in the case of M. luteus and the three others strains tested. How the recognition of M. luteus by PGRP-SA leads to the activation of Toll by cleaving Spatzle remains to be determined in molecular terms. PGRP-SA has no protease domain and it is speculated that conformational changes, induced on binding to microbial patterns, may activate associated proteases, analogous to that described for mannose-binding lectin associated proteases (MASPs) in the activation of the lectin pathway. Finally, the observation that seml mutant flies remain capable of mounting a Toll-dependent antifungal response clearly shows that the upstream mechanisms leading to activation of Toll are specific for each class of microorganisms. The versatility of the Toll-like receptors on mammalian immune-responsive cells is probably paralleled by a versatility of circulating pattern-recognition receptors in Drosophila (Michel, 2001).

Search PubMed for articles about Drosophila PGRP-SA

Bahuguna, S., Atilano, M., Glittenberg, M., Lee, D., Arora, S., Wang, L., Zhou, J., Redhai, S., Boutros, M. and Ligoxygakis, P. (2022). Bacterial recognition by PGRP-SA and downstream signalling by Toll/DIF sustain commensal gut bacteria in Drosophila. PLoS Genet 18(1): e1009992. PubMed ID: 35007276

Buchon, N., Poidevin, M., Kwon, H. M., Guillou, A., Sottas, V., Lee, B. L., Lemaitre, B. (2009). A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc. Natl. Acad. Sci. 106(30): 12442-12447. PubMed ID: 19590012

Gottar, M., et al. (2006). Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127(7): 1425-37. PubMed ID: 17190605

Michel, T., Reichhart, J. M., Hoffmann, J. A. and Royet, J. (2001). Drosophila Toll is activated by Gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414(6865): 756-759. PubMed ID: 11742401

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

date revised: 11 August 2023

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