spätzle
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)
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 Sp”tzle 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).
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).
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).
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).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
spätzle: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References
Society for Developmental Biology's Web server.