InteractiveFly: GeneBrief

persephone: Biological Overview | References

Gene name - persephone

Synonyms -

Cytological map position - 17B5-17B5

Function - enzyme

Keywords - Immune response, toll pathway, defense response to fungus

Symbol - psh

FlyBase ID: FBgn0030926

Genetic map position - X:18,378,515..18,380,954 [-]

Classification - Trypsin-like serine protease

Cellular location - secreted hemolymph protein

NCBI link: EntrezGene
psh orthologs: Biolitmine
Recent literature
Duneau, D. F., Kondolf, H. C., Im, J. H., Ortiz, G. A., Chow, C., Fox, M. A., Eugenio, A. T., Revah, J., Buchon, N. and Lazzaro, B. P. (2017). The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated Drosophila. BMC Biol 15(1): 124. PubMed ID: 29268741
This study used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. Both virgin and mated females are more susceptible than mated males to most, but not all, infections. The lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster was investigated. Females were found to have a higher number of phagocytes than males, and ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, it was found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. It was additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll pathway mutants compared to wildtype hosts highlights the key role of the Toll pathway in D. melanogaster sexual dimorphism in resistance to infection. Altogether, these data demonstrate that Toll pathway activity differs between male and female D. melanogaster in response to bacterial infection.
Patrnogic, J. and Leclerc, V. (2017). The serine protease homolog spheroide is involved in sensing of pathogenic Gram-positive bacteria. PLoS One 12(12): e0188339. PubMed ID: 29211760
In Drosophila, recognition of pathogens such as Gram-positive bacteria and fungi triggers the activation of proteolytic cascades and the subsequent activation of the Toll pathway. This response can be achieved by either detection of pathogen associated molecular patterns or by sensing microbial proteolytic activities ("danger signals"). Previous data suggested that certain serine protease homologs (serine protease folds that lack an active catalytic triad) could be involved in the pathway. This study generated a null mutant of the serine protease homolog spheroide (sphe). These mutant flies are susceptible to Enterococcus faecalis infection and unable to fully activate the Toll pathway. Sphe is required to activate the Toll pathway after challenge with pathogenic Gram-Positive bacteria. Sphe functions in the danger signal pathway, downstream or at the level of Persephone.
Nakano, S., Kashio, S., Nishimura, K., Takeishi, A., Kosakamoto, H., Obata, F., Kuranaga, E., Chihara, T., Yamauchi, Y., Isobe, T. and Miura, M. (2023). Damage sensing mediated by serine proteases Hayan and Persephone for Toll pathway activation in apoptosis-deficient flies. PLoS Genet 19(6): e1010761. PubMed ID: 37319131
The mechanisms by which the innate immune system senses damage have been extensively explored in multicellular organisms. In Drosophila, various types of tissue damage, including epidermal injury, tumor formation, cell competition, and apoptosis deficiency, induce sterile activation of the Toll pathway, a process that requires the use of extracellular serine protease (SP) cascades. Upon infection, the SP Spatzle (Spz)-processing enzyme (SPE) cleaves and activates the Toll ligand Spz downstream of two paralogous SPs, Hayan and Persephone (Psh). However, upon tissue damage, it is not fully understood which SPs establish Spz activation cascades nor what damage-associated molecules can activate SPs. In this study, using newly generated uncleavable spz mutant flies, it was revealed that Spz cleavage is required for the sterile activation of the Toll pathway, which is induced by apoptosis-deficient damage of wing epidermal cells in adult Drosophila. Proteomic analysis of hemolymph, followed by experiments with Drosophila Schneider 2 (S2) cells, revealed that among hemolymph SPs, both SPE and Melanization Protease 1 (MP1) have high capacities to cleave Spz. Additionally, in S2 cells, MP1 acts downstream of Hayan and Psh in a similar manner to SPE. Using genetic analysis, it was found ess.

In Drosophila, molecular determinants from fungi and Gram-positive bacteria are detected by circulating pattern-recognition receptors. Published findings suggest that such pattern-recognition receptors activate as-yet-unidentified serine-protease cascades that culminate in the cleavage of Spätzle, the endogenous Toll receptor ligand, and trigger the immune response. This study demonstrates that Gram-positive Specific Serine protease (Grass) defines a common activation cascade for the detection of fungi and Gram-positive bacteria mediated by pattern-recognition receptors. The serine protease Persephone, shown before to be specific for fungal detection in a cascade activated by secreted fungal proteases, was also required for the sensing of proteases elicited by bacteria in the hemolymph. Hence, Persephone defines a parallel proteolytic cascade activated by 'danger signals' such as abnormal proteolytic activities (El Chamy, 2008).

This paper reports the generation of a null mutation of the gene encoding the serine protease Grass. On the basis of RNA-mediated interference experiments, this protease has been reported before to be involved in Toll activation after infection with Gram-positive bacteria (Kambris, 2006). This study shows, by analyzing null-mutant phenotypes, that Grass was indeed involved in Toll activation after both infection with Gram-positive bacteria and fungal infection. Grass acted 'downstream' of the circulating pattern-recognition receptors (PRRs) that detect conserved cell-wall molecules of Gram-positive bacteria and fungi (peptidoglycan and β-glucan, respectively). The data further suggested that the protease Psh might sense proteolytic activities elicited by both fungal infection and infection with Gram-positive bacteria. The Toll pathway is activated by expression of the fungal protease Pr1 (Gottar, 2006) and, as shown here, by injection of proteases from B. subtilis or A. oryzea. This activation was much lower in psh mutant flies. In contrast, the Toll pathway was activated by peptidoglycans in a strictly Grass-dependent, Psh-independent way. Toll activation in response to live microorganisms involved both Grass and Psh acting in parallel pathways, whereas heat-killed fungi or bacteria did not need Psh for activation of the Toll pathway. Psh seems to sense the presence of abnormal proteolytic activities in the hemolymph. That idea was supported by the observation that considerable overexpression of Grass in transgenic flies resulted in partially Psh-dependent activation of the Toll pathway. That result suggests that Psh is able to sense the artificially high activity of Grass in the hemolymph in the same way it detects pathogen-derived proteolytic activities. It also indicates that Grass is in different configurations when artificially overexpressed in the absence of signal and when normally activated by PRR-dependent microbial recognition. In the latter case, Grass would be associated with other serine proteases or serine protease homologs, such as Spirit, Sphinx or Spheroid (Kambris, 2006), in a complex with PRRs, directing Grass activity toward SPE. In contrast, overexpressed Grass would be detected as abnormal proteolytic activity by Psh, resulting in 'downstream' activation of SPE through Psh. This finding can be correlated this finding with the phenotype resulting from mutation of the gene necrotic, which encodes a serine protease inhibitor whose inactivation leads to the deleterious and abnormal activation of several proteolytic activities, resulting in early death of mutant flies. One consequence of these abnormal proteolytic activities is constitutive Toll pathway activation. The various phenotypes associated with the necrotic mutation are all strictly Psh dependent. Indeed, Psh was isolated as a genetic suppressor of the necrotic mutation (El Chamy, 2008).

A new model is proposed for Toll activation during the immune response of Drosophila. The model proposed so far is based on two proteolytic cascades that are activated by circulating receptors able to discriminate between bacterial and fungal infection and a third cascade required for sensing fungal proteases. The results have shown that a first proteolytic cascade, which includes Grass, is activated by the binding of microbial cell-wall components to PRRs. It is further suggested that a second proteolytic cascade is activated by proteases secreted from microorganisms or abnormal proteolytic activity in the hemolymph, which are sensed by Psh in a way reminiscent of the 'guard system' in plants. This first cascade is called the 'PRR-dependent extracellular pathway'. The molecular mechanism of activation of this proteolytic cascade, which probably involves the formation of a multiprotein complex containing PRRs, is still unknown. The second cascade is called the 'danger signal extracellular pathway'. Both pathways are required for full activation of Toll-dependent immune responses against pathogens (El Chamy, 2008).

It is possible that during evolution, a proteolytic cascade system 'upstream' of the Drosophila Toll receptor provided flexibility that allowed the appearance of new detection mechanisms. The components of a protease cascade 'downstream' of GNBP1 have been purified from Tenebrio molitor, a coleopteran insect (Kim, 2008). However, none of those components shows strong homology to Grass, which suggests that the proteolytic cascades involved in immune defense are subjected to divergent evolution. Psh may have evolved secondarily to add a new level of defense by sensing the activity of invading microorganisms. It is suspected that the detection of bacteria and fungi was first based on specific recognition of molecular patterns. In the 'arms race' between host and pathogens, and given the probable emergence of escape mechanisms in pathogens masking their cell-wall components or hampering detection, Psh provided a way of sensing microorganisms indirectly by their activity. Being aware of proteolytic activity allows the detection of microorganisms, as many bacteria and fungi excrete proteases during the invasion process. The proteases they secrete, such as Pr1, provide the host with early signs of infection, and sensing of the proteases by Psh enables a rapid response against invaders. Detection of microbial activity seems more flexible than recognition of conserved molecular patterns during host-pathogen interaction. Indeed, the finding that psh shows the highest polymorphism among Drosophila species (Jiggins, 2007) shows that psh is under strong selection (El Chamy, 2008).

Several virulence factors of E. faecalis are proteases that probably target Psh, as heat-killed bacteria do not require Psh for Toll pathway activation. E. faecalis is not a natural Drosophila pathogen, as it must be artificially introduced into flies. The identification of proteases from Gram-positive entomopathogenic bacteria is crucial for understanding the host-pathogen interaction. Gram-negative bacteria and bacilli strongly engage the IMD pathway but also moderately and transiently activate the Toll pathway. The possibility that some proteases secreted by Gram-negative bacteria could activate the Toll pathway through Psh is still open (El Chamy, 2008).

Pathogen sensing by a dual system comprising a first branch that recognizes molecules common to many classes of microbes (pathogen-associated molecular patterns) and a second branch that responds to virulence factors, either directly or indirectly through their effects on host targets ('danger signal'), is well described in the plant immune system. This study has shown that a similar dual system is at work in Drosophila. Mammalian cells use PRRs, such as Toll-like receptors (which sense extracellular microbial determinants) and Nod-like receptors (which sense intracytoplasmic microbial determinants; Fritz, 2006), to detect microorganisms. It has been suggested that 'danger signals' such as virulence factors or endogenous proteins released by damaged cells may also be detected directly by Toll-like receptors or Nod-like receptors. In addition, some pathogens secrete proteases, which allows them to degrade adherent junctions and penetrate the epithelial barrier. Some bacterial proteases are able to cleave protease-activated receptor 2 (PAR2), which leads to the secretion of antimicrobial peptides and inflammatory cytokines in epithelial cells. PARs are G protein-coupled transmembrane receptors that are activated by cleavage of their own amino-terminal domain, which acts as a tethered ligand. Activation of PARs by endogenous proteases of the thrombin and trypsin families leads to inflammatory responses by means of the NF-kappaB, AP1 and c/EBP-β transcription factors. Cleavage of PARs by injury-activated thrombin or bacterial proteases seems to be a 'danger signal'-sensing mechanism very similar to Psh-dependent activation of the Toll pathway in flies. These results demonstrate that as in plants, sensing of 'danger signals' works together with the detection of pathogen-associated molecular patterns in animals. Analysis of a dual system encompassing PARs or other as-yet-unidentified sensors in parallel with PRRs will undoubtedly shed new light on the understanding of microbial sensing in mammals (El Chamy, 2008).

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 GNBP3hades have distinct phenotypes as regards Toll pathway activation and because the double mutant psh; GNBP3hades 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 GNBP3hades 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).

Activation of Drosophila Toll During Fungal Infection by a Blood Serine Protease

Drosophila host defense to fungal and Gram-positive bacterial infection is mediated by the Spaetzle/Toll/cactus gene cassette. It has been proposed that Toll does not function as a pattern recognition receptor per se but is activated through a cleaved form of the cytokine Spaetzle. The upstream events linking infection to the cleavage of Spaetzle have long remained elusive. This study reports the identification of a central component of the fungal activation of Toll. Ethylmethane sulfonate-induced mutations in the persephone gene, which encodes a previously unknown serine protease, block induction of the Toll pathway by fungi and resistance to this type of infection (Ligoxygakis, 2002).

The Drosophila host defense is a multifaceted process, which involves the challenge-dependent synthesis of potent antimicrobial peptides by the fat body, a functional equivalent of the mammalian liver. Two intracellular signaling pathways mediate the synthesis of these peptides: the Toll and the Imd pathway. Toll is activated during fungal and Gram-positive bacterial defenses, and the Imd pathway is predominantly activated by Gram-negative bacterial infections. Toll was initially identified as a gene that controls the establishment of dorsoventral polarity in early embryogenesis. A proteolytically cleaved form of the cysteine-knot growth factor Spaetzle (Spz) functions as the extracellular ligand of Toll in both embryonic development and the immune response. During embryonic patterning, Spz cleavage is achieved by the sequential activation of three serine proteases required in the germ line: Gastrulation defective, Snake, and Easter. However, in null mutants of these proteases, challenged-induced activation of the Toll pathway is not affected, as illustrated by wild-type induction of the antifungal peptide drosomycin. The implication is that infection may activate some other protease(s) that cleaves Spz to its active form. Immune-induced cleavage of Spz by blood serine proteases is conceptually similar to blood coagulation or complement activation, where inappropriate activation is prevented by the action of serine protease inhibitors (serpins). Flies mutant for the blood serpin Necrotic (Nec) exhibit a constitutively activated antifungal host defense, and Spz is constitutively cleaved in this mutant background (Levashina, 1999). The nec pleiotropic phenotypes include spontaneous melanization, cellular necrosis, and death in early adulthood. These changes probably all reflect a role of the nec serpin gene in controlling activation of one or more proteolytic cascades (Ligoxygakis, 2002).

To identify components of the antifungal response cascade that activate Spz, the first chromosome was screened for ethylmethane sulfonate-induced suppressors of the nec phenotypes. From 9700 mutagenized male flies trans-heterozygous for nec, five suppressors were isolated that belong to the same complementation group. This suppressor mutation was called persephone (psh). Mutations in psh suppressed all nec phenotypes. In particular, psh;nec double mutants displayed a life-span comparable to that of wild-type (WT) flies, showed no spontaneous melanization, and did not express drosomycin in a constitutive manner. When challenged with fungi, psh mutants exhibited a severely reduced level of drosomycin transcription as compared with WT flies. Induction of drosomycin by Gram-positive bacteria was at WT levels in psh mutants. Finally, expression of diptericin, which is controlled by the Imd pathway, was not affected following Gram-negative bacterial infection (Ligoxygakis, 2002).

It was further noted that psh flies were highly susceptible to fungal infections, behaving in this respect as Toll pathway mutants. Conversely, psh flies showed a WT pattern of survival after immune challenge by Gram-positive bacteria. As expected, psh flies were resistant to Gram-negative bacterial infection, which activates the Imd pathway (Ligoxygakis, 2002).

For epistasis studies, fly lines were used overexpressing a cleaved form of Spz through the UAS/GAL4 system. These flies exhibit a challenge-independent expression of drosomycin. The levels of drosomycin transcription after overexpression of spz were notably similar in psh mutants and in WT flies. This result indicates that the psh mutation inactivates a gene upstream of spz (Ligoxygakis, 2002).

With a deficiency kit spanning the first chromosome, the psh mutation was mapped to the chromosomal region 17A-17B. This region contains two serine protease genes, CG6361 and CG6367. The genomic sequence of the CG6367 protease in psh5 was compared with that of WT flies and noted the transition of a G nucleotide to an A in the sequence encoding the conserved 'serine signature' sequence (Gly-Asp-Ser-Gly-Gly-Pro), which results in a Gly to Glu change at position 340. In the sequence of psh1 and psh4, a transition of a C to a T was observed, which is expected to result in the change of the His of the catalytic triad to a Tyr at position 187 (Ligoxygakis, 2002).

To ensure that these mutations were responsible for the observed phenotypes in psh mutants, rescue experiments were undertaken using the UAS/GAL4 system with the female fat body-specific yolkGAL4 as a driver. Itis noted that UAS-CG6367/yolkGAL4 flies constitutively express drosomycin. Furthermore, it was observed that overexpression of the CG6367 protease restores the ability of psh flies to respond to fungal infection. Neither an activated form of Easter nor the CG6361 protease contained in the deficiency that uncovers the psh locus were able to rescue the observed sensitivity to fungi. These results indicate that CG6367 is the serine protease responsible for the psh phenotype (Ligoxygakis, 2002).

The deduced sequence of the PSH protein indicates the presence of a putative signal peptide (amino acids 1 to 20), suggesting that the protein is secreted and present in the hemolymph, as has been shown for Nec. Transfer of hemolymph from nec flies to WT flies carrying a drosomycin-GFP (drs-GFP) reporter resulted in expression of drs-GFP. In contrast, transfer of hemolymph from psh;nec flies did not induce drosomycin expression. This indicates that the blood-borne factor responsible for Toll activation observed in nec hemolymph transfer is suppressed in psh;nec flies. Finally, transfer of hemolymph from flies overexpressing WT psh (UAS-CG6367/daughterlessGAL4) to drs-GFP flies induced challenge-independent expression of drosomycin. These results confirm the presence of immune-responsive components of the system in the hemolymph (Ligoxygakis, 2002).

The blood serine protease Persephone is the first identified component of the cascade, which was hypothesized to activate Toll following an immune challenge. Serine proteases are initially synthesized as inactive zymogens containing an NH2-terminal prodomain and a COOH-terminal catalytic domain. Activation requires proteolytic cleavage of the zymogen at a defined site by a specific activating protease or a nonenzymatic ligand. The sequence of PSH also predicts an NH2-terminal prodomain. This prodomain contains a CLIP module most homologous to those in Easter, Snake, the horseshoe crab proclotting factor, and the Bombyx mori prophenoloxidase-activating enzyme. Thus, common organizing principles may direct hemolymph clotting, immune response, and developmental serine protease cascades in arthropods. psh is the first described mutation to specifically impair Toll-dependent induction of drosomycin by fungal infection in Drosophila without affecting Gram-positive bacterial induced responses. Mutations have been reported recently that affect activation of the Toll pathway by Gram-positive bacteria and activation of the Imd pathway by Gram-negative bacteria. The mutated genes encode members of the family of soluble or membrane proteins referred to as peptidoglycan recognition proteins (PGRPs), in reference to their initial discovery as Gram-positive interacting proteins. Toll activation by Gram-positive bacteria is mediated by a soluble PGRP, whereas that of Imd by Gram-negative infection involves a putative membrane PGRP. Taken together, the results on the psh mutation and those on mutations in the soluble PGRP-SA and the putative membrane PGRP-LC now define three distinct upstream pathways mediating response to fungal infections and to infections by Gram-positive or Gram-negative bacteria. These data set the stage for a detailed analysis of the events leading from recognition of infection to activation of intracellular signaling pathways and consequent transcription of appropriate groups of genes concurring to fight the respective infections. Whereas PGRPs can be considered as bona fide pattern recognition receptors, psh has no microbial pattern recognition-binding domain. It is anticipated that an as-yet unidentified, upstream fungal pattern recognition receptor functions to activate the protease function of psh (Ligoxygakis, 2002).

Functions of Persephone orthologs in other species

Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta

Proteolytic activation of phenoloxidase (PO) and the cytokine Spatzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. This study presents biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta. Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. This study found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR(176)*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH(112)*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpatzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, this study has elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar (Wang, 2020).


Search PubMed for articles about Drosophila Persephone

Bischoff, V., et al. (2004). Function of the drosophila pattern-recognition receptor PGRP-SD in the detection of Gram-positive bacteria. Nat. Immunol. 5: 1175-1180. PubMed ID: 15448690

Brown, G. D. and Gordon, S. (2001). Immune recognition. A new receptor for beta-glucans. Nature 413: 36-37. PubMed ID: 11544516

El Chamy, L., Leclerc, V., Caldelari, I. and Reichhart, J. M. (2008). Sensing of 'danger signals' and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of Toll. Nat. Immunol. 9(10): 1165-70. PubMed ID: 18724373

Fritz, J. H., Ferrero, R. L., Philpott, D. J. and Girardin, S. E. (2006). Nod-like proteins in immunity, inflammation and disease. Nat. Immunol. 7: 1250-1257. PubMed ID: 17110941

Gobert, V., et al. (2003). Dual activation of the Drosophila toll pathway by two pattern recognition receptors. Science 302(5653): 2126-30. PubMed ID: 14684822

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

Ferrandon, D., Imler, J. L. and Hoffmann, J. A. Sensing infection in Drosophila: Toll and beyond. Semin. Immunol. 16: 43-53. PubMed ID: 14751763

Jang, I. H., et al. (2006). A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity. Dev. Cell 10: 45-55. PubMed ID: 16399077

Jiggins, F. M. and Kim, K. W. (2007). A screen for immunity genes evolving under positive selection in Drosophila. J. Evol. Biol. 20: 965-970. PubMed ID: 17465907

Kambris, Z., et al. (2006). Drosophila immunity: a large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation. Curr. Biol. 16: 808-813. PubMed ID: 16631589

Kaneko, T. and Silverman, N. (2005). Bacterial recognition and signalling by the Drosophila IMD pathway. Cell Microbiol. 7: 461-469. PubMed ID: 15760446

Kaneko, T., et al. (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, C. H., et al. (2008). A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced Toll pathway in an Insect. J. Biol. Chem. 283: 7599-7607. PubMed ID: 18195005

Kim, Y. S., et al. (2000). Gram-negative bacteria-binding protein, a pattern recognition receptor for lipopolysaccharide and beta-1,3-glucan that mediates the signaling for the induction of innate immune genes in Drosophila melanogaster cells. J. Biol. Chem. 275: 32721-32727. PubMed ID: 10827089

Lee, S. H., et al. (2006). Effects of solutes on solubilization and refolding of proteins from inclusion bodies with high hydrostatic pressure. Protein Sci. 15: 304-313. PubMed ID: 16385003

Levashina, E. A., et al. (1999). Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science 285(5435): 1917-9. PubMed ID: 10489372

Ligoxygakis, P., Pelte, N., Hoffmann, J. A. and Reichhart, J. M. (2002). Activation of Drosophila Toll during fungal infection by a blood serine protease. Science 297: 114-116. PubMed ID: 12098703

Ma, C. and Kanost, M. R. (2000). A beta1,3-glucan recognition protein from an insect, Manduca sexta, agglutinates microorganisms and activates the phenoloxidase cascade. J. Biol. Chem. 275: 7505-7514. PubMed ID: 10713054

Ochiai, M. and Ashida, M. (2000). A pattern-recognition protein for beta-1,3-glucan. The binding domain and the cDNA cloning of beta-1,3-glucan recognition protein from the silkworm, Bombyx mori. J. Biol. Chem. 275: 4995-5002. PubMed ID: 10671539

Wang, Y., Yang, F., Cao, X., Zou, Z., Lu, Z., Kanost, M. R. and Jiang, H. (2020). Hemolymph protease-5 links the melanization and Toll immune pathways in the tobacco hornworm, Manduca sexta. Proc Natl Acad Sci U S A 117(38): 23581-23587. PubMed ID: 32900946

Biological Overview

date revised: 2 October 2009

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