Protein Interactions

Ventral activation of a transmembrane receptor, Toll, forms a crucial step in dorsoventral axis establishment in Drosophila embryos. The ventral ligand for Toll seems to be a proteolytic fragment of the Spätzle protein (Roth, 1994).

A soluble, extracellular factor has been found to induce ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, a 24 x 10(3) M(r)protein that has polarizing activity has been purified. The purified protein is recognized by antibodies to theC-terminal half of the Spätzle protein, indicating that this polarizing activity is a product of thespätzle gene. The purified protein is smaller than the primary translation product of spätzle,suggesting that proteolytic processing of Spätzle on the ventral side of the embryo is required togenerate the localized, active form of the protein (Schneider, 1994).

Twelve maternal effect genes (the dorsal group and cactus) are required for the establishment ofthe embryonic dorsal-ventral axis in the Drosophila embryo. Embryonic dorsal-ventral polarity isdefined within the perivitelline compartment surrounding the embryo by the ventral formation of aligand for the Toll receptor. By transplantation of perivitelline fluid thepresence of three separate activities present in the perivitelline fluid can be shown to restore dorsal-ventralpolarity to mutant easter, snake, and spätzle embryos, respectively. These activities are not capableof defining the polarity of the dorsal-ventral axis; instead they restore structures according to theintrinsic dorsal-ventral polarity of the mutant embryos. They appear to be involved in the ventralformation of a ligand for the Toll protein. This process requires serine proteolytic activity; theinjection of serine protease inhibitors into the perivitelline space of wild-type embryos results in theformation of dorsalized embryos (Stein, 1992).

Proteolytic activation of Spatzle requires the sequential action of four different members of thetrypsin family. The first protease in this pathway is encoded bynudel, which is expressed in the somatic follicle cells of the ovary (which secrete the eggshell), whereas the other three proteases are expressed by the germlinecells. gastrulation defective (gd) is closely related to the trypsin family, but the protein encoded by gd lacks a number of amino acid residues crucial for protease activity, so its biochemical function is not clear. Downstream of gd is the protease encoded by snake, which acts upstream of Easter, the final protease known in this pathway. The Easter protease is likely to be the direct proteolytic activator of Spatzle (Misra, 1998 and references).

The sequential activities of these four members of the trypsin family of extracellular serine proteases are required for the production of the ventrally localized ligand that organizes the dorsal-ventral pattern of the Drosophila embryo. The last protease in this sequence, encoded by easter, is a candidate to activate proteolytically the ligand encoded by spatzle. Processed Easter is present in extremely low amounts in the early embryo making it difficult to detect. Nevertheless, proteolytic processing of a catalytically inactivated Easter can be detected in vivo. Thus proteolytically activated Easter can be detected only in mutants of easter that lack catalytic activity and not in wild type. Activated Easter is rapidly converted into a high molecular mass complex, which may contain a protease inhibitor. The complex has a molecular mass of 80 to 85 kDa, larger than the 50 kDa Easter zymogen. The high molecular mass band (which is called Ea-X because the nature of the protein complexed with Easter is unknown) is not present in embryos that lack easter mRNA, so it appears to be a novel form of the Easter protein. The Ea-X complex is very stable; it is not disrupted by boiling in SDS for 30 minutes (Misra, 1998).

The Ea-X complex is shown to contain the proteolytically processed Easter protein. Based on the distribution of cysteines in the wild-type Easter protein, the N-terminal pro-domain is expected to remain disulfide bonded to the C-terminal catalytic domain after zymogen activation. If the activated Easter in Ea-X is a two-chain protease with the pro-and catalytic domains associated by a disulfide bond, then wild-type Ea-X should migrate more slowly on non-reducing gels (where the pro-domain would remain associated with the complex), than on reducing gels, which would contain only the Easter catalytic domain and X. Under non-reducing conditions, Ea-X migrates more slowly than the mutant EaN-X (coded for by an N-terminal deletion mutant of easter in which the pro-domain is deleted and the signal sequence is fused directly to the catalytic domain), confirming that the wild-type Ea in Ea-X has been proteolytically cleaved at the zymogen activation site anddemonstrating that the pro-domain of the wild-type enzyme remains disulfide-bonded to the catalytic domain after zymogen cleavage (Misra, 1998).

Easter zymogen activation is controlled by a negative feedback loop from Dorsal, the transcription factor at the end of the signaling pathway. Mutations that block the intracellular signaling pathway leading to the activation of Dorsal do not block the formation of Ea-X. In fact, all these downstream mutants contain 3- to 5-fold more Ea-X than wild-type embryos. Even mutant embryos that lack spatzle mRNA and protein show increased levels of Ea-X, indicating that Ea-X is not a stable complex of Easter, with its putative substrate, Spatzle. In ventralized cactus mutant embryos, the amount of Ea-X is less than half the amount present in wild-type embryos. Since the amount of Ea-X reflects the amount of activated Easter, these results suggest that a feedback loop regulated by nuclear Dorsal acts back across the plasma membrane of the syncytial embryo to regulate activation of the Easter zymogen. The accumulation of Ea-X in downstream mutants during the early zygotic phase occurs at the same time that signaling through the pathway occurs. While the zymogen (unprocessed) form of Easter is present at fertilization, Ea-X does not appear until 1 hour after fertilization, at approximately the time that the Easter protein is active in the embryo and increased during the next 2 hours of development. As soon as Ea-X is detectable, there is more Ea-X in Toll mutants than there is in wild-type embryos. Thus, well before the final gradient of Dorsal is achieved, at about 2.5 hours after fertilization, the level of Easter processing appears to be modified by this feedback loop (Misra, 1998).

The most likely molecules to bind the active site are substrates and inhibitors. Because Ea-X is extremely stable and because Ea-X does not include Spatzle, its likely substrate, it is hypothesized that Ea-X is an inhibited complex and that the other component of Ea-X is an inhibitor. A serine protease inhibitor could play an important role in patterning by preventing the spread of Easter activity away from its initial site of activation. The Ea-X complex has the properties expected for a complex with a protease inhibitor of the serpin family. The serpins are a family of proteins that act as suicide substrates. Serpin suicide substrates are inhibitors that are cleaved by their target enzymes. Serpins interact with the serine that constitutes the activesite of serine proteases, thereby irreversibly inactivating the protease. Most serpins are 40-60 kDa in size, which would be consistent with the size difference between the predicted masses of the catalytic domains of Easter and Ea-X. As with other serpin-protease complexes, Ea-X formation requires the active site serine and the protease-bindingpocket. Also, like other serpin complexes, the Ea-X complex is extremely stable. Only a single cloned Drosophila serpin-encoding gene has been well characterized; this serpin is transferred from the male to the female during copulation and its function is not known. Additional serpins have been identified in other insects, making it very likely that Drosophila will have a number of different serpin-encoding genes. The reverse genetic approach of purifying the high molecular mass complex, microsequencing and gene cloning will ultimately make it possible to test whether the other component of Ea-X is a serpin or another protease inhibitor (Misra, 1998).

The dorsal-ventral asymmetry of the Drosophila embryo is established by a signal that is transmitted through the uniformly distributed Toll receptor, resulting in a graded relocation of Dorsal into the nuclei of the blastoderm embryo. gastrulation defective is one of the earliest acting of the maternally required genes that is crucial to this process. The gastrulation defective gene has been cloned and characterized and found to encode a protein structurally related to the serine protease superfamily, which also includes the Snake, Easter, and Nudel proteins. These data provide additional support for the involvement of a protease cascade in generating an asymmetric signal (i.e., asymmetric Spatzle activity) during the establishment of dorsal-ventral polarity in the Drosophila embryo (Konrad, 1998).

Injection experiments involving the use of dominant active Easter and Snake, as well as injection of perivitelline (PV) fluid from dorsal mutant embryos into gd mutant embryos, leads to production of ventral elements at the site of injection, rather than in the normal ventral region. These data suggest that D/V polarity is established by asymmetric presentation of the Toll ligand to the oocyte. PV fluid from dorsal mutant embryos (thought to be depleted of Spätzle ligand because of the presence of the Toll receptor) can rescue D/V polarity in snake and easter mutant embryos. This same PV fluid cannot restore normal ventral structures to either gd or nudel embryos. In contrast, injection of PV fluid from Toll mutant embryos (thought to contain active Spätzle ligand) into gd embryos produces ventral structures at the site of injection. The same fluid injected into snake or easter embryos produces embryos with normal polarity, independent of the site of injection. This result strongly suggests that Gd is a key component required for establishing the localized activation of Spätzle and thus the asymmetric activation of Toll. Because the temperature-sensitive period for both Nudel and Gd action includes a period before fertilization when the initial D/V asymmetry is known to be established , it is possible that these two gene products cooperate to form a localized anchor for a Spätzle activating complex (Konrad, 1998 and references).

Easter and Snake both share significant structural homology with extracellular trypsin-like serine proteases. Experiments using dominant active forms of Snake and Easter show that Snake activates Easter, which in turn activates the Spätzle ligand. In combination with the somatically expressed genes, windbeutel, pipe, and nudel, Gd activates Snake in a location-dependent manner that marks the future ventral cells. The recent cloning of nudel indicates that Ndl is a large (350 kDa) extracellular glyco-protein with motifs suggesting that it might bind to extracellular matrix as well as to other proteins. Nudel also contains a serine protease catalytic domain. The occurrence of several protease-like proteins both upstream (Nudel) and downstream (Snake, Easter) of Gd may be responsible for the multiply processed Gd peptides observed on Western blots. The physical location of the Nudel protein (including whether it is incorporated as a component of the vitelline membrane) is not yet known, although the fragility of the ndl embryos suggests that Ndl may be required for stability of the vitelline membrane. GD mRNA is expressed in follicle cells beginning at about stage 10 and may be graded in a ventral to dorsal manner, although it is expressed uniformly in the nurse cell/oocyte complex. The location of Gd remains to be determined. Four of the five currently identified genes needed for asymmetric activation of the Spz ligand encode secreted members of the serine protease superfamily. It is proposed that Gd functions as part of an anchored complex that triggers a proteolytic activation hierarchy involving Nudel, Snake, and Easter, resulting in localized activation of Spz ligand and asymmetry along the D/V axis (Konrad, 1998 and references).

The Toll family of receptors is required for innate immune response to pathogen-associated molecules, but the mechanism of signaling is not entirely clear. In Drosophila the prototypic Toll regulates both embryonic development and adult immune response. The host protein Spatzle can function as a ligand for Toll because Spatzle forms a complex with Toll in transgenic fly extracts and stimulates the expression of a Toll-dependent immunity gene, drosomycin, in adult flies. Constitutively active mutants of Toll form multimers that contain intermolecular disulfide linkages. These disulfide linkages are critical for the activity of one of these mutant receptors, indicating that multimerization is essential for the constitutive activity. Furthermore, systematic mutational analysis revealed that a conserved cysteine-containing motif, different from the cysteines used for the intermolecular disulfide linkages, serves as a self-inhibitory module of Toll. Deleting or mutating this cysteine-containing motif leads to constitutive activity. This motif is located just outside the transmembrane domain and may provide a structural hindrance for multimerization and activation of Toll. Together, these results suggest that multimerization may be a regulated, essential step for Toll-receptor activation (Hu, 2004)

Gastrulation Defective is a serine protease involved in activating the receptor Toll to polarize the Drosophila embryo

The products of four maternal genes, in addition to snake and easter, namely gastrulation defective (gd), nudel (ndl), pipe (pip), and windbeutel (wind), are required for processing of Spätzle to the Toll ligand. In the genetically ordered pathway for production of the Toll ligand, these genes act upstream of the snake gene and therefore are presumed to encode components required to activate the Snake protease. According to recent studies, the Pipe and Windbeutel proteins function during oogenesis to generate a spatially localized factor that defines the ventral side of the embryo. In current models, this factor is hypothesized to direct the ventral production of the Toll ligand by spatially restricting the activation of Snake and Easter, perhaps via a mechanism involving the Nudel and GD proteins. Nudel, a large modular protein with a central serine protease domain, is functionally complex and apparently involved in several processes important for embryogenesis. The Nudel protease is required for cross-linking of the eggshell, in addition to its role in establishing the embryonic dorsoventral axis. This observation has raised the intriguing possibility that the Nudel protease is involved in creating an extracellular matrix structure necessary for activating the proteolytic cascade that produces the Toll ligand (Han, 2000).

The role of the GD protein in activating this proteolytic cascade has been particularly enigmatic. GD is suspected to have an important role in this process, in part because it has the structure of a regulated serine protease like Snake and Easter, with a C-terminal domain homologous to serine proteases. However, it has been unclear whether GD functions as a serine protease because of peculiarities of its primary structure, such as the absence of a catalytic serine in the conserved position, or a recognizable proteolytic cleavage site for zymogen activation. It also has been unclear whether GD functions during early embryogenesis, rather than oogenesis, and is therefore directly involved in activating the proteolytic cascade that produces the Toll ligand. In addition, the position of GD with respect to Nudel, Pipe, and Windbeutel in the pathway producing the Toll ligand has not been known. In this study the GD protein is shown to be a serine protease. Three other genes act to restrict GD activity to the ventral side of the embryo. The data support a model in which the GD protease catalyzes the ventral activation of the proteolytic cascade that produces the Toll ligand (Han, 2000).

The fact that gd mutant embryos can be rescued by injection of GD mRNA indicates that the GD protein can function during early embryogenesis and thus could have a direct role in producing the Toll ligand. The downstream proteases Snake and Easter likely function in the perivitelline space of the embryo. It is presumed that GD is also a component of this extracellular compartment, since it has a signal sequence and is efficiently secreted by transfected S2 cells. However, it was not possible to rescue mutant embryos by injecting the perivitelline space of gd mutant embryos with recombinant GD in S2 cell culture medium, perhaps because of an insufficient amount of protein that also precluded extensive biochemical analysis (Han, 2000).

Genetic as well as biochemical evidence has been provided that GD functions as a serine protease. (1) The biological activity of GD RNA is destroyed by site-directed mutagenesis of putative catalytic serine and aspartic acid residues in GD. (2) A 29-kDa proteolytic fragment of recombinant GD protein reacts in vitro with affinity reagents specific for active serine proteases. The affinity labeling experiments also suggest that GD is synthesized as a zymogen that becomes activated by proteolytic cleavage, because a proteolytic fragment of GD, but not the full-length protein, reacts with the affinity reagents. GD lacks a recognizable zymogen activation site, so where cleavage normally occurs for GD to become an active serine protease is unclear. An active protease was generated by digestion of GD by trypsin, which is specific for basic residues, implying that GD could be normally activated by cleavage after a basic residue (Han, 2000).

The identification of GD as a serine protease raises the question, what is GD's substrate? A possible candidate is Snake, which is immediately downstream of GD in the Toll signaling pathway. The Snake zymogen may be activated by cleavage after a leucine residue and therefore by a chymotrypsin-like enzyme that prefers large hydrophobic residues. Whether GD has this specificity is unclear, since it diverges in sequence from other serine proteases in the region containing certain key residues that determine amino acid specificity. The fact that the 29-kDa form of GD reacts with affinity reagents designed for trypsin-like enzymes might suggest that GD is specific for basic residues. However, chymotrypsin also can cleave after basic residues, albeit inefficiently, and therefore could react with these affinity reagents under favorable conditions. Clearly, further biochemical experiments will be required to discern the amino acid specificity of the GD protease and to determine whether GD directly activates Snake (Han, 2000).

The RNA injection experiments reveal an important relationship between GD activity and Toll signaling. Injection of embryos with high concentrations of GD RNA causes more embryonic cells than normal to adopt ventral and lateral fates. A similar result has been observed in analogous experiments with spätzle RNA and with RNAs encoding 'preactivated' Snake and Easter but not the zymogen forms of these proteases. Thus, the level of GD activity may determine the amount of Spätzle processed to the Toll ligand, presumably by controlling the level of active Snake and Easter. In this case, an important function of Snake and Easter could be to amplify the signal generated by GD into the appropriate amount of Toll ligand necessary to establish the embryonic dorsoventral axis (Han, 2000).

These experiments revealed that gd acts downstream of nudel, pipe, and windbeutel, because the normal activity of each of these three genes is not required for injected GD RNA to induce ventral and lateral structures. After injection with a high level of GD RNA, embryos lacking maternal activity of nudel, pipe, or windbeutel develop a lateralized phenotype, which lacks the dorsoventral asymmetry of the ventralized phenotype seen when wild-type and gd mutant embryos are similarly injected. Thus, GD activity appears to be spatially uniform in the absence of normal Pipe, Windbeutel or Nudel function, implying that these three proteins are required to restrict GD activity to the ventral side of the embryo (Han, 2000).

How is GD activated? Although Nudel is the only known protease in the Toll signaling pathway that acts genetically upstream of GD, the data suggest that Nudel is not essential for GD protease activity. GD could be activated by an unidentified protease; alternatively, GD could self-activate. Proteases that initiate the protease cascades of blood clotting and apoptosis have a low level of enzymatic activity as zymogens, but become fully active upon a conformational change or upon oligomerization induced by a nonenzymatic cofactor. If GD is activated by a similar mechanism, then it should have a low level of protease activity as a zymogen (presumably below the sensitivity of detection in these affinity labeling experiments). In this case, the high concentration of GD protein produced by RNA injection into the embryo may have led to spontaneous GD activation that mimics and bypasses the normal activation mechanism. A model consistent with available data posits that the Nudel protease activates a cofactor, perhaps the ventral determinant provided by Pipe and Windbeutel, which in turn is necessary for activating the GD protease (Han, 2000).

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).

Regulation of Easter activity is required for shaping the Dorsal gradient in the Drosophila embryo

Dorsoventral polarity of the Drosophila embryo requires maternal spätzle-Toll signaling to establish a nuclear gradient of Dorsal protein. The easter gene encodes a serine protease that generates processed Spätzle, which in turn acts as the Toll ligand. The shape of the Dorsal gradient is altered in embryos produced by females carrying dominant alleles of easter (eaD) that do not require cleavage of the zymogen for activity. By examining the expression domains of the zygotic genes zen, sog, rho and twist, which are targets of nuclear Dorsal, it has been shown that the slope of the Dorsal gradient is progressively flattened in stronger eaD alleles. In the wild-type embryo, activated Easter is found in a high molecular weight complex called Ea-X, which is hypothesized to contain a protease inhibitor. In eaD embryo extracts, an Easter form corresponding to the free catalytic domain is detected; this form is never observed in wild type. Mutant eaD proteins retain protease activity, as determined by the production of processed Spätzle both in the embryo and in cultured Drosophila cells. These experiments suggest that the eaD mutations interfere with inactivation of catalytic Easter, and imply that this negative regulation is essential for generating the wild-type shape of the Dorsal gradient (Chang, 2002).

The production of the C-terminal catalytic domain has been observed only with catalytically inactive easter mutants, exemplified by the ea8 allele, a missense mutation located in the presumptive substrate binding pocket, and the eaS338A allele, in which the active site serine-338 was replaced with alanine (Misra, 1998). It was hypothesized that the C-terminal catalytic domain in the eaD alleles, in contrast to the recessive mutations, retain protease activity after zymogen activation. In order to test this model, EaD protease activity was assessed in two different experiments (Chang, 2002).

(1) The level of processed Spätzle was determined in eaD embryo extracts, because Easter protease activity generates processed Spätzle. No processed Spätzle is produced in ea- extracts; a higher level of processed Spätzle is observed in a transformant line carrying the eaDeltaN mutation. Extracts were prepared from embryos laid by eaD/+ and eaD/ea- females. Precursor and processed forms of Spätzle were detected on an immunoblot probed with antibodies specific to the Spätzle C-terminal domain. As expected, a lower level of processed Spätzle is observed in the +/ea- embryo extract compared with the wild-type embryo extract. Similarly, the level of processed Spätzle is lower in the eaD/ea- embryo extract compared with the corresponding eaD/+ embryo extract. In general, the amount of processed Spätzle in each of the eaD/ea- lanes appears roughly comparable with the level in the +/ea- lane. The exception to this generalization is observed with the lateralizing ea5.13 allele. The amount of processed Spätzle observed in extracts prepared from embryos laid by ea5.13/+ and ea5.13/ea- females is reproducibly higher than in wild type (Chang, 2002).

(2) An injection assay was used to ask whether wild-type and eaD activated Easter could be detected in the embryo. By transferring perivitelline fluid from one embryo to another, Stein (1991) characterized a ventralizing activity exhibiting properties expected of the Toll ligand. When this 'polarizing activity', later identified to be processed Spätzle, was injected into the perivitelline space of recipient embryos laid by pipe- females, the site of injection defined the ventral pole of a new axis (Stein, 1991; Stein, 1992; Schneider, 1994). It was reasoned that by using donor embryos laid by spz- females (and thereby removing the presumptive Toll ligand), it might be possible to detect the activity responsible for generating processed Spätzle, i.e. active Easter (Chang, 2002).

Perivitelline fluid transfer experiments were carried out using gastrulating donor embryos laid by different mutant females; perivitelline fluid was transferred into stage 4 recipient embryos laid by pipe- females. No axis-inducing activity was detected from donor embryos laid by either spz- or spz- Toll- females, suggesting that active Easter is either rapidly inactivated or sequestered in a non-transplantable complex. Furthermore, no activity was detected from donor embryos laid by ea83l spz- females. By contrast, axis-inducing activity was observed from donor embryos laid by ea5.13 spz- females. This observation not only provides functional evidence for Ea5.13 protease activity, but also demonstrates that the activity remains stable until gastrulation, many hours after zymogen activation (Chang, 2002).

In order to study the Spätzle cleavage reaction, precursor Spätzle and wild-type EaDeltaN proteins were co-expressed in cultured Drosophila S2 cells. Production of processed Spätzle was observed in the conditioned medium, with the level of cleavage dependent on the amount of EaDeltaN expressed. No formation of the inhibited EaDeltaN-X form was observed in these transfected S2 cells (Chang, 2002).

The effect of the ea83l and ea5.13 mutations on the cleavage reaction was analyzed. The level of Spätzle processing carried out by Ea83lDeltaN was indistinguishable from wild-type EaDeltaN, suggesting that the ea83l mutation does not affect Easter catalytic activity. By comparison, Ea5.13DeltaN exhibits weaker protease activity, although it appears to be expressed at higher levels. This result suggests that the lateralized phenotype produced by the ea5.13 mutation arises from two separate effects: (1) lack of proper inhibition following zymogen activation leads to loss of the dorsal zen domain, and (2) reduced Easter catalytic activity prevents formation of the ventral twist domain (Chang, 2002).

Drosophila immunity: A large-scale in vivo RNAi screen identifies five serine proteases required for Toll activation

Unlike mammalian Toll-like receptors, the Drosophila Toll receptor does not interact directly with microbial determinants but is rather activated upon binding a cleaved form of the cytokine-like molecule Spatzle (Spz). During the immune response, Spz is thought to be processed by secreted serine proteases (SPs) present in the hemolymph that are activated by the recognition of gram-positive bacteria or fungi. In the present study, an in vivo RNAi strategy was used to inactivate 75 distinct Drosophila SP genes. This collection was then screened for SPs regulating the activation of the Toll pathway by gram-positive bacteria. This study reports the identification of five novel SPs that function in an extracellular pathway linking the recognition proteins GNBP1 and PGRP-SA to Spz. Interestingly, four of these genes are also required for Toll activation by fungi, while one is specifically associated with signaling in response to gram-positive bacterial infections. These results demonstrate the existence of a common cascade of SPs upstream of Spz, integrating signals sent by various secreted recognition molecules via more specialized SPs (Kambris, 2006).

Until recently, only one SP, Psh, has been shown to act upstream of Toll in response to fungi. Via a large-scale RNAi screen, five novel SPs have been identified that regulate Toll activity in response to gram-positive bacterial infections. Three of them, Spirit (for Serine Protease Immune Response Integrator), Grass, and SPE, are functional chymotrypsin-like SPs containing a Clip domain N-terminal to the catalytic domain. The Clip domain is exclusively found in insect SPs and is believed to play a regulatory role in the sequential activation of SPs. For instance, it is present in Psh as well as in Snake and Easter, two SPs that participate in the processing of Spz during embryonic development. In contrast to spirit and SPE (Spatzle Processing Enzyme), grass (Gram-positive Specific Serine protease) is required to resist gram-positive bacterial but not fungal infection, and its knock-down induces a phenotype similar to those induced by GNBP1 or PGRP-SA mutations. This is in agreement with a model in which Grass is activated by GNBP1 and PGRP-SA after recognition of gram-positive peptidoglycan. Grass then transmits a signal that is integrated by a common core of downstream SPs including Spirit and SPE. In agreement with a position of Grass upstream in the Toll-activating cascade, the expression of an activated form of SPE and Spirit but not Grass can trigger the antimicrobial peptide gene Drosomycin (Drs) in S2 cells. Further signal amplification could occur since both spirit and SPE are induced during the immune response in a Toll pathway-dependent manner. Thus, this combination of controlled proteolytic SPs activation and transcriptional positive feedback would ensure an adequate response to infection (Kambris, 2006).

Surprisingly, two SPs identified in in the screen, Spheroide and Sphinx1/2, are unlikely to possess proteolytic activity, because their protease-like domain lacks the catalytic serine residue. This class of SP homologs (SPHs) represents one quarter of Drosophila SP-related proteins and is thought to have regulatory functions. Several SPHs have been shown to be involved in the proteolytic cascade that regulates the cleavage of prophenoloxidase in other insects, supporting a role of this class of SP-related proteins during the immune response. Since knock-downs of spheroide and sphinx1/2 induces the same phenotype as that of SPE and spirit, it is suggested that these two SPHs may act as adaptors or regulators of SPE and Spirit, possibly by localizing the two SPs in close proximity to Spz and/or the fat-body cell membrane to promote robust activation of the Toll receptor (Kambris, 2006).

RNAi of the five SPs did not affect activation of melanization nor did it suppress lethality induced by the inactivation of spn27A. In agreement with two recent studies, RNAi of SP7 (CG3066) reduces melanization at the wound site, confirming the implication of this SP in the prophenoloxidase cascade. The RNAi of this SP did not affect Toll activation by gram-positive bacteria. Collectively, these data indicate that distinct SPs mediate melanization and Toll activation, underlining the complexity of SP cascades regulating the Drosophila immune response. Further studies, including the generation of null mutations, are required to analyze in more detail the function of these five SPs upstream of Toll (Kambris, 2006).

This study represents the first extensive in vivo RNAi screen of a large gene family in Drosophila. A key advantage of this strategy for functional genomic studies is that genes required during development can be inactivated in a tissue- and temporal-specific manner. It is especially suitable for the analysis of genes encoding secreted proteins such as SPs that function in a non-cell-autonomous manner and therefore require in vivo studies. The SP-RNAi fly collection described in this study and made available to the scientific community should pave the way for additional biochemical studies and genetic screens to further decipher the signaling pathways acting upstream of Toll as well as to identify additional important SP functions (Kambris, 2006).

The Toll receptor was originally identified as an indispensable molecule for Drosophila embryonic development and subsequently as an essential component of innate immunity from insects to humans. Although in Drosophila the Easter protease processes the pro-Spätzle protein to generate the Toll ligand during development, the identification of the protease responsible for pro-Spätzle processing during the immune response has remained elusive for a decade. This study reports a protease, called Spätzle-processing enzyme (SPE), that is required for Toll-dependent antimicrobial response. Flies with reduced SPE expression show no noticeable pro-Spätzle processing and become highly susceptible to microbial infection. Furthermore, activated SPE can rescue ventral and lateral development in embryos lacking Easter, showing the functional homology between SPE and Easter. These results imply that a single ligand/receptor-mediated signaling event can be utilized for different biological processes, such as immunity and development, by recruiting similar ligand-processing proteases with distinct activation modes (Jang, 2006).

An important question is how SPE itself is activated during the immune response. SPE does not appear to be activated by Snake, the protease that activates Easter; SPE must be preactivated to rescue ventral and lateral development in embryos lacking Easter; moreover, the SPE zymogen is not cleaved by Snake in vitro. One candidate to be a direct activator of SPE is the protease encoded by psh, which is required to activate Toll signaling in response to fungal infection (Ligoxygakis, 2002b) and, this study has shown, is also required to process SPE into an active protease. However, the SPE zymogen is not cleaved by the Psh protease when coexpressed in human embryonic kidney cells (data not shown), suggesting that another protease directly activates SPE. While the direct activator of SPE still needs to be identified, this work has nonetheless defined a new protease cascade involving Psh and SPE with a role in immunity. Since SPE is also activated during the immune response to G+ bacterial infection, which does not involve psh, there may exist another protease cascade in which SPE is activated. The possible existence of two protease cascades that converge on SPE as a terminal protease provides for a versatile immune system in which the same signaling pathway that activates a potent immune response can be used for defense against distinct pathogens. Fungal and G+ bacterial infection also appears to trigger the activation of another cascade in which the terminal protease, pro-PO activating enzyme (PPAE), activates a key enzyme in the melanization reaction that encapsulates pathogens. The activation of two different immune responses involving SPE and PPAE by a common trigger would be an advantageous mechanism for enhancing host survival after microbial infection (Jang, 2006).

Protease cascades have diverse biological roles in vertebrates and invertebrates, ranging from digestive processes to fertilization, immunity, development, and tissue remodeling. This work highlights the functional relationships between protease cascades involved in distinct processes such as embryonic development and innate immunity. SPE and Easter are the terminal proteases of two different protease cascades involved in development and immunity, yet both process the Spätzle protein to activate the Toll signaling pathway. The essential difference between SPE and Easter appears to be that they require distinct mechanisms for activation, which allows the Toll signaling pathway to be activated in response to different triggers and thus used in very different physiological processes. Another link between development and immunity is provided by Spn27A, a serine protease inhibitor that regulates both Easter and the pro-PO cascade. Interestingly, Spn27A does not appear to regulate SPE, as evidenced by the fact that Toll signaling is not constitutively activated in flies mutant for Spn27A. Thus, although SPE and Easter possess common substrate specificity and similar enzymatic activity, the striking regulatory differences between SPE and Easter in terms of their activation/inhibition mode may confer dual physiological functions on the Spätzle-Toll signaling cassette. These structural and functional relationships between the protease cascades involved in Drosophila development and immunity support the idea that an ancestral protease cascade gave rise to those with diverse functions in present day organisms (Jang, 2006).

A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. This study purified and cloned a novel upstream serine protease of SPE that was named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway (Kim, 2008).

This study describes three novel findings regarding the Lys-type PG recognition Toll signaling pathway: 1) biological functions of a novel Tm-SAE have been determined; 2) the sequential molecular activation mechanisms of three SPs involved in the Toll signaling pathway have been determined; and 3) the first biochemical evidence of how the Tm-PGRP-SA-mediated Lys-type PG recognition signal is transferred to Spz, leading to antimicrobial activity in vivo, is provided. Based on these results, it is proposed that this PGRP-SA/GNBP1/MSP/SAE/SPE/Spz cascade may be an essential unit that triggers the Lys-type PG recognition signaling pathway in response to Gram-positive bacteria infection in insects (Kim, 2008).

These results do not seem to correspond with those recently reported by Kambris (2006). They suggested that two Drosophila catalytic SPs (Dm-Grass and Dm-Spirit) and two non-catalytic SP homologs (SPHs), such as Dm-Spheroide and Dm-Sphinx1/2, are involved in the PG-dependent Drosophila Toll pathway. By homology research with known SPs, it turned out that Dm-Grass and Dm-Spirit are clip domain-containing trypsin-like SPs. Dm-Spheroide and Dm-Sphinx1/2 each have a non-catalytic SP domain but no clip domains at the N terminus. They also have Gly and Ile residues, respectively, instead of a Ser residue in the catalytic site of SPs. The reason why this study did not identify similar SPs and SPHs is unclear, and further studies are necessary to answer this question. However, one plausible explanation for this can be that Tenebrio SPHs may exist with serpins in the hemolymph and are not directly involved in the Toll pathway activation. A horseshoe crab factor D, a SPH-like molecule in Limulus, was co-purified with a horseshoe crab serpin, suggesting that SPH might make a complex with serpins in vivo. By performing RNAi experiments against Drosophila SPs or SPHs, there is a possibility that serpins can be released to the hemolymph by lack of SPH and that catalytic SPs, such as Dm-Grass and Dm-Spirit might be trapped by the released serpins leading to inhibition of the Toll pathway activation (Kim, 2008).

Similarly, the current data may provide a clue for the screening of Dm-MSP and Dm-SAE-like Drosophila counterparts in the Toll pathway. A plausible candidate for a Dm-MSP is the protease encoded by CG31217, which has the same domain organization as Tenebrio and Tribolium MSPs. Therefore, a valuable study would be one which addresses whether flies with reduced CG31217 expression can transfer Lys-type PG recognition signals to Dm-SPE or whether they will become susceptible to a Gram-positive bacteria infection. However, when the amino acid residues of the tentative cleavage site and the substrate specificity pocket residues of the CG31217 protease were examined, their amino acid residues were quite different from those of beetle MSPs. Notably, the CG31217 zymogen has a putative chymotrypsin-like cleavage site between Phe-368 and Ser-369 and the elastase-like substrate specificity pocket residues of CG31217 protease were identified as Leu-557 (c189), Ala-593 (c216), and Thr-604 (c226). These data suggest that there is a possibility that an SP other than the CG31217 protease may function as an initial SP in the Drosophila Toll pathway. If the current model can be applied to the Drososphila system, the direct downstream SP of the CG31217 protein may have the same cleavage site as the CG31217 protease, since it was shown for the Tm-MSP and Tm-SAE zymogens. 24 different Drosophila clip-domain-containing SPs were found in a Blast search of the Drosophila genome sequence. To find out which of these 24 Drosophila clip domain SPs correspond to the Tm-SAE, further genetic analysis studies are necessary. However, the possibility cannot be excluded that as yet unidentified isoforms of Dm-SAE and Dm-MSP-like protease might induce the activation of an alternative Toll pathway in Drosophila (Kim, 2008).

In summary, biochemical studies shed further light on the molecular mechanism of how the Lys-type PG recognition signal is transferred to the Toll receptor. The work supports a model in which a PG recognition complex activates three different SPs zymogens sequentially. This three-step proteolytic cascade-dependent processing of the extracellular protein Spz and then the binding of the processed Spz to Toll receptor are required for the induction of antimicrobial peptides expression in this innate immune pathway. A greater understanding of this cascade will also facilitate the development of a kit to rapidly and sensitively detect bacterial PG in blood and food products (Kim, 2008).

Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component

The insect Toll signaling pathway is activated upon recognition of Gram-positive bacteria and fungi, resulting in the expression of antimicrobial peptides via NF-kappaB-like transcription factor. This activation is mediated by a serine protease cascade leading to the processing of Spätzle, which generates the functional ligand of the Toll receptor. Three serine proteases have been discovered that mediate Toll pathway activation induced by lysine-type peptidoglycan of Gram-positive bacteria. However, the identities of the downstream serine protease components of Gram-negative bacteria binding protein 3 (GNBP3), a receptor for a major cell wall component beta-1,3-glucan of fungi, and their order of activation have not been characterized yet. This study identified three serine proteases that are required for Toll activation by beta-1,3-glucan in the larvae of a large beetle, Tenebrio molitor. The first one is a modular serine protease functioning immediately downstream of GNBP3 that proteolytically activates the second one, a Spätzle-processing enzyme-activating enzyme that in turn activates the third serine protease, a Spätzle-processing enzyme. The active form of Spätzle-processing enzyme then cleaves Spätzle into the processed Spätzle as Toll ligand. In addition, it was shown that injection of beta-1,3-glucan into Tenebrio larvae induces production of two antimicrobial peptides, Tenecin 1 and Tenecin 2, which are also inducible by injection of the active form of Spätzle-processing enzyme-activating enzyme or processed Spätzle. These results demonstrate a three-step proteolytic cascade essential for the Toll pathway activation by fungal beta-1,3-glucan in Tenebrio larvae, which is shared with lysine-type peptidoglycan-induced Toll pathway activation (Roh, 2009).

Recent genetic studies using either in vivo RNAi strategy or loss-of-function mutation have demonstrated that several Drosophila serine proteases, such as Persephone, Grass, and Spirit, function upstream of the Drosophila SPE during activation of Toll (Kambris, 2006). Furthermore, El Chamy (2008) demonstrated that Drosophila Grass functions as a signaling component required for both the detection of fungi by GNBP3 and the sensing of Gram-positive bacteria by peptidoglycan recognition protein PGRP-SA. Also, it was suggested that another Drosophila serine protease, Persephone, previously shown to be specific for fungal detection, was required for the sensing of proteases that are released by fungi and bacteria (El Chamy, 2008). This indicates that Drosophila Persephone defines a parallel proteolytic cascade activated by virulence factors in the hemolymph. However, Drosophila Grass and Persephone genes do not show any homology with Tenebrio MSP, which functions as an apical serine protease common to both Tenebrio GNBP3 and PGRP-SA/GNBP1 complex. These results suggest the existence of significant differences in the proteolytic cascades working upstream of Toll in Tenebrio and Drosophila. In addition to this diversity of serine proteases, the gene expression sites of Tenebrio AMPs are also different from those of Drosophila AMPs. The Tenebrio Tenecin 1 gene was strongly induced in hemocytes of the Tenebrio larvae, while Drosophila AMPs are mainly produced by the fat body. Taken together, the differences in the organization of the serine protease cascade, in the type of AMPs and in the site of AMP synthesis between Drosophila and Tenebrio could represent adaptation mechanisms to fight specific environmental pathogens or may be linked to differences in their physiology. Analyzing differences and similarities in the mechanisms used by insects to fight microbial infection should shed light on the evolution of the immune system in this important phylum (Roh, 2009).

The activated Tenebrio SPE converted both the 79-kDa Tenebrio pro-phenoloxidase and clip-domain serine protease homologue 1 zymogen to their matured forms to generate an active melanization complex. This complex, consisting of a 76-kDa Tenebrio-activated phenoloxidase and a 43-kDa serine protease homologue 1, efficiently produces melanin on the surface of bacteria, and this activity has a strong bactericidal effect. Because Tenebrio SPE also participates in the activation of the Toll pathway by GNBP3 upon β-1,3-glucan sensing and by PGRP-SA upon Lys-type PG recognition, these results indicate that both the melanization and the Toll pathway immune modules are sharing a common serine protease cascade for the regulation of these two major innate immune responses (Roh, 2009).

In summary, these biochemical studies shed light on the molecular mechanism regulating the Toll pathway by fungi. This work supports a model in which bacterial Lys-type PG and β-1,3-glucan recognition activate a common proteolytic cascade involving three different serine protease zymogens that are sequentially processed. This three-step proteolytic cascade leads to the maturation of Spätzle and the activation of the Toll intracellular signaling that control the synthesis of AMPs. A greater understanding of these two cascades could also facilitate the development of novel kits to detect bacteria and fungi in clinical and food products rapidly and sensitively (Roh, 2009).

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 (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. 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 (Gottar, 2006). 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. 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. 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. 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).

Role for sumoylation in systemic inflammation and immune homeostasis in Drosophila larvae

To counter systemic risk of infection by parasitic wasps, Drosophila larvae activate humoral immunity in the fat body and mount a robust cellular response resulting in encapsulation of the wasp egg. Innate immune reactions are tightly regulated and are resolved within hours. To understand the mechanisms underlying activation and resolution of the egg encapsulation response and examine if failure of the latter develops into systemic inflammatory disease, parasitic wasp-induced changes in the Drosophila larva were correlated with systemic chronic conditions in sumoylation-deficient mutants. It has been reported that loss of either Cactus, the Drosophila (IkappaB) protein, or lesswright/Ubc9, the SUMO-conjugating enzyme, leads to constitutive activation of the humoral and cellular pathways, hematopoietic overproliferation and tumorogenesis. This study reports that parasite infection simultaneously activates NF-kappaB-dependent transcription of Spätzle processing enzyme (SPE) and cactus. Endogenous Spätzle protein (the Toll ligand) is expressed in immune cells and excessive SPE or Spätzle is pro-inflammatory. Consistent with this function, loss of Spz suppresses Ubc9- defects. In contrast to the pro-inflammatory roles of SPE and Spätzle, Cactus and Ubc9 exert an anti-inflammatory effect. Ubc9 maintains steady state levels of Cactus protein. In a series of immuno-genetic experiments, the existence of a robust bidirectional interaction between blood cells and the fat body was demonstrated, and it is proposed that wasp infection activates Toll signaling in both compartments via extracellular activation of Spätzle. Within each organ, the IkappaB/Ubc9-dependent inhibitory feedback resolves immune signaling and restores homeostasis. The loss of this feedback leads to chronic inflammation. These studies not only provide an integrated framework for understanding the molecular basis of the evolutionary arms race between insect hosts and their parasites, but also offer insights into developing novel strategies for medical and agricultural pest control (Paddibhatla, 2010).

Parasitic wasps are a large group of insects that typically attack other insects. Because of the absolute dependence on their insect hosts, parasitic wasps are of enormous commercial interest and can replace insecticides to control insect pests. The motivation of this study was to gain a clearer understanding of how insect larvae respond to attacks of these natural enemies. Using an immuno-genetic approach in Drosophila, this study found that the same Toll-dependent NF-kappaB mechanism that rids Drosophila of microbial infections also defends the host against metazoan parasites. However, because of critical differences in their size and mode of entry, the combination of immune responses summoned in the two cases is different. While phagocytosis and systemic humoral responses (the latter originating from the fat body and in the gut) are the principal mechanisms of host defense against bacteria and fungi, the development of parasitic wasp eggs is blocked primarily by encapsulation response (Paddibhatla, 2010).

Data is presented that demonstrate the critical requirement of the humoral arm in both the activation and resolution of egg encapsulation. The bi-directional interaction between the blood cells and the fat body occurs via cell non-autonomous effects of SPE/Spz, where these secreted proteins synthesized in one compartment can activate immune signaling in the other. Recent reports corroborate a signaling role for Spz derived from blood cells in the expression of antimicrobial peptides from the larval fat body in response to microbes. Because activation/deactivation of both immune arms is accomplished via the IkappaB/Ubc9-dependent feedback loop that has both, cell autonomous and cell non-autonomous effects, it is proposed that this shared mechanism allows efficient coordination between the immune organs and helps restore normal immune homeostasis within the infected host (Paddibhatla, 2010).

The mechanism that coordinates the activation and resolution of both immune arms after parasite infection involves a balance between the positive (SPE) and negative (Cactus) components. Infection induces nuclear localization of Dorsal and Dif, and the transcription of both SPE (which resolves over time) and cactus (transcription levels off). This Cactus-dependent regulation is essential for the downregulation of SPE transcription and the termination of the encapsulation response. The negative feedback loop of Cactus in flies is similar to the one identified for IkappaBα in mammalian cells (Paddibhatla, 2010).

In Ubc9 mutants, the stability of Cactus protein is compromised, and Toll signaling persists during the extended larval life. Accordingly, knockdown of Cactus in blood cells (Hml>cactusRNAi) promotes inflammation, aggregation and melanization. It is proposed that loss of immune homeostasis leads to constitutive SPE expression and activation of Spätzle, which promotes the development of chronic inflammation. Thus, sumoylation serves an anti-inflammatory function in the fly larva (Paddibhatla, 2010).

This study has identified at least two distinct biological roles of sumoylation: first, an essential role in blood cells, where the post-translational modification curbs proliferation in the lymph gland in the absence of infection. This conclusion is also strongly supported by restoration of normal hematopoietic complement in mutants expressing wild type Ubc9 only within a limited lymph gland population. Second, sumoylation is essential to sustain significant, steady state levels of Cactus. In mammalian cells, sumoylation of IkappaBα protects it from antagonistic, ubiquitination-mediated degradation. The results are consistent with the mammalian model where Cactus sumoylation would be expected to modulate its half-life (Paddibhatla, 2010).

Cytokine activation and function are hallmarks of the normal inflammatory response in mammals. A key finding of this study is that active Spz serves a pro-inflammatory function in fly larvae. This first report of any pro-inflammatory molecule in the fly confirms that cytokines activate inflammation across phyla. As with mammalian cytokines that act as immuno-stimulants, Spz is expressed, and is therefore likely to activate the blood cells surrounding the parasite capsule. Active Spz promotes blood cell division, migration and infiltration much like high levels of Dorsal and Dif, suggesting that the cell biological changes triggered by SPE/Spz are mediated by target genes of Dorsal and Dif. It is intriguing that the integrity of the basement membrane (as visualized by Collagen IV expression pattern) appears to be important for orchestrating blood cells to the site of 'diseased self' (the mutant fat body in this study) in a manner that may be similar to recognition of the non-self parasitic egg, underscoring the parallel roles of basement membrane proteins in the origin and development of inflammation in both flies and mammals (Paddibhatla, 2010).

Although excessive (active) Spz is proinflammatory, its loss leads to reduction in the hematopoietic complement. For example mutants lacking spz (spzrm7/spzrm7) exhibit a 40% reduction in circulating blood cell concentration and these animals do not encapsulate wasp eggs as efficiently as their heterozygous siblings. These observations suggest that active Spz's normal proliferative/pro-survival functions, required for maintaining the normal hematopoietic complement, are fundamentally linked to its immune function for the activation and recruitment of blood cells to target sites. Thus, the autocrine and paracrine hematopoietic and inflammatory effects of Spz are amplified in the presence of hyperactive Toll receptor, excessive Dorsal/Dif, or the loss of Cactus/Ubc9 inhibition, resulting in production of hematopoietic tumors. It is possible that mutations in other, unrelated, genes that yield similar inflammatory tumors arise due to the loss of Toll-NF-kappaB dependent immune homeostasis (Paddibhatla, 2010).

These results highlight the central role of the Dorsal/Dif proteins not only in immune activation, but also in the resolution of these responses. Proteomic studies have confirmed that Dorsal is a bona fide SUMO target and its transcriptional activity is affected by sumoylation. Dorsal and Dif exhibit genetic redundancy in both the humoral and cellular responses. It is possible that this redundancy ensures that immune reactions against microbes and parasites are efficiently resolved to allow proper host development (Paddibhatla, 2010).

In nature, parasitic wasps are continually evolving to evade or suppress the immune responses of their hosts. To this end, they secrete factors or produce protein complexes with specific molecular activities to block encapsulation. These studies provide the biological context in which the effects of virulence factors produced by pathogens and parasites on primordial immune pathways can be more clearly interpreted. The molecular identity of wasp factors which actively suppress humoral and cellular responses (e.g., those in L. heterotoma remains largely unknown. Such virulence factors are likely to be 'anti-inflammatory' as they clearly interfere with host physiology that ultimately disrupts the central regulatory immune circuit defined in these studies (Paddibhatla, 2010).

Encapsulation reactions of non-self (wasp egg) or diseased self tissues (fat body) of the kind in the Drosophila larva are not only reported in other insects, but the reaction is likely to be similar to mammalian granulomas, which are characterized by different forms of localized nodular inflammation. Furthermore, the phenotypes arising from persistent signaling in mutants recapitulate the key features of mammalian inflammation: i.e., reliance on conserved signaling mechanism, the requirement for cytokines, and sensitivity to aspirin. These studies also reveal a clear link between innate immunity and the development and progression of hematopoietic cancer in flies, as has been hypothesized from work in mammalian systems. In the past, genetic approaches in Drosophila have served well to dissect signaling mechanisms governing developmental processes in animals. The fly model with hallmarks of acute and chronic mammalian inflammatory responses will provide deep insights into signaling networks and feedback regulatory mechanisms in human infections and disease. It can also be used to test the potency and mechanism of action of pesticides, anti-inflammatory and anti-cancer agents in vivo (Paddibhatla, 2010).

Persephone/Spatzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila

Apoptosis is an evolutionarily conserved mechanism that removes damaged or unwanted cells, effectively maintaining cellular homeostasis. It has long been suggested that a deficiency in this type of naturally occurring cell death could potentially lead to necrosis, resulting in the release of endogenous immunogenic molecules such as DAMPs (damage-associated molecular patterns) and a non-infectious inflammatory response. However, the details about how danger signals from apoptosis-deficient cells are detected and translated to an immune response are largely unknown. This study found that Drosophila mutants deficient for Dronc, the key initiator caspase required for apoptosis, produced the active form of the endogenous Toll ligand Spatzle (Spz). It is speculated that, as a system for sensing potential DAMPs in the hemolymph, the dronc mutants constitutively activate a proteolytic cascade that leads to Spz proteolytic processing. It was demonstrated that Toll signaling activation required the action of Persephone, a CLIP-domain serine protease that usually reacts to microbial proteolytic activities. These findings show that the Persephone proteolytic cascade plays a crucial role in mediating DAMP-induced systemic responses in apoptosis-deficient Drosophila mutants (Ming, 2014).

spätzle: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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