InteractiveFly: GeneBrief

serpin-27A : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

In arthropods, the melanization reaction is associated with multiple host defense mechanisms leading to the sequestration and killing of invading microorganisms. Arthropod melanization is controlled by a cascade of serine proteases that ultimately activates the enzyme prophenoloxidase (PPO: in Drosophila PPO is called Black cells), which, in turn, catalyzes the synthesis of melanin. A Drosophila serine protease inhibitor protein, Serpin-27A regulates the melanization cascade through the specific inhibition of the terminal protease (prophenoloxidase-activating enzyme). Serpin-27A is required to restrict the phenoloxidase activity to the site of injury or infection, preventing the insect from excessive melanization (De Gregorio, 2002b). Evidence is presented for the coordination of hemolymph-borne melanization with activation of the Toll pathway in the Drosophila host defence (De Gregorio, 2002a; Ligoxygakis, 2002). The melanization reaction requires Toll pathway activation and depends on the removal of the Drosophila serine protease inhibitor Serpin27A. Flies deficient for this serpin exhibit spontaneous melanization in larvae and adults. Microbial challenge induces its removal from the hemolymph through Toll-dependent transcription of an acute phase immune reaction component (Ligoxygakis, 2002).

Insects have a sensitive mechanism for identifying pathogens and an array of strategies for defending themselves against microbial attack. To combat infection, the fruit fly relies on both constitutive and inducible defense mechanisms (Tzou, 2002). However, the first line of defense that prevents microbial invasion into the hemocoel is structural. It is comprised of the external cuticle, the gut peritrophic matrix, and the tracheal lining. When pathogens breach these barriers, they activate a wide range of inducible reactions. Perforation of the cuticle by injury or by microbial infection rapidly activates proteolytic cascades that lead to blood coagulation and melanization. Upon subsequent microbial or parasitic infection, a cellular immune response, mediated by different hemocyte types, is mounted and participates in pathogen/parasite clearance by phagocytosis or encapsulation. During systemic infection, a large set of inducible effector molecules, such as antimicrobial peptides (AMP), stress-responsive proteins, and other factors required for opsonization and iron sequestration, are secreted into the hemolymph (Tzou, 2002; De Gregorio, 2002b).

Apart from the regulation of AMP gene expression, very little is known about the other defense mechanisms that combat infection in Drosophila. The most dramatic and immediate immune response in Drosophila is the melanization reaction observed at the site of cuticular injury or on the surface of pathogens, parasites, and parasitoids invading the hemocoel. The blackening of the wounded area of the cuticle or the surface of invading organisms results from the de novo synthesis and deposition of melanin. In arthropods, melanization plays an important role in defense reactions, such as wound healing, encapsulation, sequestration of microbes, and the production of toxic intermediates, that are speculated to kill invading microorganisms (Ashida, 1998; Söderhäll, 1998). So far, no regulators of the melanization cascade have been identified at the molecular level in the fruit fly (De Gregorio, 2002b).

Although there is a paucity of information concerning the Drosophila melanization reaction, this response has been investigated in more detail in other arthropods (Söderhäll, 1998). It requires the activation of phenoloxidase (PO) (EC 1.14.18.1), an enzyme that catalyses the oxidation of mono- and diphenols to orthoquinones, which are then polymerized nonenzymatically to melanin. Insect phenoloxidase has been found to exist in the hemolymph plasma and the cuticle as an inactive form, called prophenoloxidase (PPO). Enzymatically inactive PPO is cleaved into active PO by a serine protease known as prophenoloxidase-activating enzyme (PPAE). PPAE also exists as an inactive zymogen that is activated through a stepwise process involving other serine proteases. PPAEs have been isolated from different insect species and other arthropods (Jiang, 1998; Lee, 1998; Satoh, 1999; Wang, 2001). Several studies indicate that the melanization cascade is triggered by injury or by recognition of microbial cell wall components, such as peptidoglycan, ß-1,3 glucan, and lipopolysaccharide (LPS), through pattern recognition proteins (Yoshida, 1996; Yu, 1999; Ma, 2000; Ochiai, 1999). Consequently, the PPO cascade is an efficient nonself-recognition system in invertebrates, and its immediate activation suggests that it may regulate aspects of the immune response other than PPO activation (De Gregorio, 2002b).

Although vertebrates do not possess an equivalent of a PPO system, the PPO cascade in insects is reminiscent of the blood clotting reactions in vertebrates. Both reactions involve serine protease cascades that must be tightly regulated to avoid a systemic activation, which is often fatal. One class of serine protease regulators ubiquitous among plants, animals, and viruses is serpins (Irving, 2000). Serpins compose a superfamily of proteins that fold into a conserved structure and employ a suicide substrate-like inhibitory mechanism (Ye, 2001). In the carboxyl terminal, they have a 30–40-residue-long 'reactive center loop' (RCL), which is exposed at the surface of protein that binds and covalently links to the active site of the target serine protease (Lawrence, 2000; Potempa, 1994). After cleavage of the RCL region, the P1 residue of the serpin forms a covalent acyl bond with the active site serine residue of the target protease (Cohen, 1977; Potempa, 1994). Like humans, fruit flies possess a high number of genes encoding serpins, which reflects the high number of serine protease genes found in this insect. However, the serpin gene family is poorly studied in Drosophila. To date, a mutation in only one serpin gene, spn43Ac, has been studied in relation to the necrotic phenotype (Levashina, 1999). The loss-of-function mutation in spn43Ac leads to constitutive activation of the Toll-mediated immune response, indicating that Spn43Ac inhibits a serine protease that functions upstream of the Toll ligand Spaetzle (De Gregorio, 2002b).

The identification of genes that regulate the melanization cascade in Drosophila has been complicated by the high number of serpin and serine protease candidate genes in the fly genome. Recently, a DNA microarray analysis helped in the selection of 5 serpin genes among the 30 encoded by the Drosophila genome that, like spn43Ac, are significantly upregulated in response to infection and might control one of the serine protease cascades associated to fly immune reactions (De Gregorio, 2001). Biochemical and genetic characterization of one of them, serpin-27A (spn27A), demonstrates that the protein encoded by this gene regulates the melanization cascade through the specific inhibition of PPO processing by the terminal serine protease PPAE (De Gregorio, 2002b).

Spn27A, like the antimicrobial peptides, is synthesized in the fat body tissue and secreted in the hemolymph. Biochemical assays show that recombinant Spn27A inhibits the activation of PPO in different insect species including Drosophila, pointing to a remarkable conservation of the melanization mechanisms. This finding is consistent with the sequence homology of PPO genes from the same species and allowed for an investigation of the role of Spn27A in heterologous systems for which the melanization cascade is better characterized than in Drosophila. The similarities between the PPO cleavage site and the reactive center loop of Spn27A prompted a test to see whether prophenoloxidase-activating enzyme PPAE was the target protease of Spn27A. Indeed recombinant Spn27A, but not a recombinant variant mutated in the reactive center loop, inhibits the PPAE purified from H. diomphalia. In agreement with the current models of serpin-serine protease interaction, the inhibition occurs through the formation of a specific covalent link between the two proteins (De Gregorio, 2002b).

These findings strongly suggest that, in Drosophila, the role of Spn27A is to modulate PPO processing through the inhibition of a still-unidentified PPAE. The role of Spn27A in melanization is supported by genetic analysis. spn27A¹ mutant flies frequently exhibit melanotic tumors and darkening of the cuticle, which may be the consequence of natural injury during the fly lifecycle. The most dramatic phenotype of spn27A¹ is, however, the excessive melanization reaction observed after injury or wasp infection. These observations indicate that the role of Spn27A is to limit the PPO cascade at the site of injury to prevent systemic melanization. The acute phase expression profile of spn27A in response to septic injury is probably critical for a tight spatial and temporal regulation of PPO activity (De Gregorio, 2002b).

The observation that some of the spn27A-deficient mutants do not present any form of constitutive melanization indicates the existence of multiple levels of regulation of the melanization reaction. An Spn27A-independant mechanism may limit the activation of PPO in the absence of stimulus. Although this has not been firmly demonstrated in Drosophila, this step is probably regulated by the availability of PPO in the hemolymph. At least in Drosophila, hemolymphatic PPO is localized in a hemocyte cell type called the crystal cell, which belongs to the oenocytoid lineage of hemocytes, known for their capacity to synthesize PPO. Crystal cells can be freely circulating in the hemolymph or sessile. Sessile crystal cells have been shown to be grouped in densely packed clusters along with plasmatocyte type hemocytes, in direct contact with the cuticular epithelial layer (Lanot, 2000). In response to different stimuli, like integumental injury or microbial invasion, crystal cells rupture and release their PPO content into the hemolymph plasma, especially around the area where stimulation occurs. The PPO may then be subjected to cleavage by PPAE in a process tightly regulated by Spn27A (De Gregorio, 2002b).

The melanization reaction is considered as an important facet of the insect host defense. Melanotic encapsulation can prevent completion of the malaria parasite lifecycle in Anopheles gambiae (Collins, 1986), while, in Drosophila, it plays an important role against infection by parasitoid wasps (Vass, 2000). However, little is known about the exact role of melanization during Drosophila immune response. The Black cells (Bc) mutation affects crystal cells and blocks the melanization reaction in the hemolymph but does not alter the inducibility of anti-microbial peptide genes. Interestingly, this mutation induces a higher susceptibility to microbial infection when combined with mutations affecting the Toll and Imd pathways. An experiment, showing that both Bc and spn27A¹ mutants are more susceptible to infection by the entomopathogenic fungus B. bassiana, indicates that a tight control of melanization is required for a proper antifungal response. The analysis of Bc and spn27A¹ mutants clearly shows that the terminal components of the melanization reaction (PPO, PPAE, and Spn27A) are not involved in the regulation of the antimicrobial peptide response. Hence, the PPO cascade and anti-microbial peptides provide two uncoupled defense systems that cooperate to fight microbial infection (De Gregorio, 2002b).

In mammals, a variety of proteolytic cascades, including blood coagulation, fibrinolysis, inflammation, and complement activation are regulated by serpins. In addition, serpins are also involved in various kinds of physiological processes, such as angiogenesis, apoptosis, neoplasia, and viral pathogenesis (Silverman, 2001). Several serpin genes have been inactivated by targeted mutation in the mouse. Some of these mutations have failed to reveal an overt phenotype, whereas others show various physiological and developmental alterations (Silverman, 2001). Spn27A and Spn43Ac (Necrotic) are two serpins characterized by genetic studies in Drosophila (Green, 2000; Levashina, 1999; De Gregorio, 2002b). Interestingly the phenotype induced by the deletion of these serpins shows similarities and differences. Both spn27A¹ and necrotic mutations result in a similar pleiotropic phenotype: female sterility, low viability at the pupal stage, and ectopic melanization in unchallenged animals. In addition, these two genes are both induced upon infection and are mainly regulated by the Toll pathway. Mutation in spn43Ac, however, induces a constitutive activation of the Toll pathway (Levashina, 1999), whereas mutation in spn27A causes an uncontrolled melanization reaction. Biochemical study clearly indicates a difference in target specificity between these two serpins: recombinant Spn43Ac is unable to inhibit the PPO cascade. It would be interesting to know whether the phenotype of each serpin mutant is mediated by more than one target protease. The observation that Bc is epistatic to (functions downstream of) spn27A indicates that the pleiotropic phenotype of spn27A¹ mutation can be explained by the multiple functions of melanization reaction in Drosophila. The role of Spn27A in early embryogenesis, which is not Bc dependent, remains to be investigated (De Gregorio, 2002b).

Since the Drosophila genome encodes five serine proteases for every serpin, it is very likely that serpins can inhibit multiple target proteases. Microarray analysis has shown that about 19 serine proteases are upregulated upon infection in Drosophila (De Gregorio, 2001). Among them, new targets of Spn27A and Spn43Ac activities might be identified (De Gregorio, 2002b).

The discovery of a regulator of the PPO pathway in Drosophila opens the way for the molecular characterization of the insect melanization cascade that plays a central role in host defense. The dramatic phenotype of spn27A¹ mutant flies underlines the critical role of serpin molecules in the tight regulation of proteolytic cascades activating host defense reactions. Furthermore, the high number of serine protease and serpin genes encoded in the fruit fly genome suggests that Drosophila is a remarkable model to understand the role of proteolytic cascades in the integrated host defense (De Gregorio, 2002b).

REGULATION

Characterization of functional activity

Interestingly, close examination of the deduced amino acids revealed that Spn27A contains a C-terminal region that is homologous to the region around the conserved cleavage site of insect prophenoloxidases (PPO). Serine proteases have been shown to cleave the propeptide of PPO between Arg and Phe within the NRFG motif to generate active PO for melanin synthesis. Furthermore, the cleavage sites of PPO from most insects and Spn27A are very similar (NRFG versus NKFG, respectively). It is hypothesized that both these proteins serve as substrates for a common serine protease. To investigate the possible involvement of Spn27A in regulating PPO activation, a PPO activation assay was performed using Drosophila pupal homogenate in the presence of recombinant Spn27A (rSpn27A). Time-course analysis shows that PO activity in the pupal homogenate increases over time, reaching a plateau after 30 min. The addition of rSpn27A to the pupal homogenates completely blocks PO activity, regardless of incubation time. Given that insect PPO activation cascades are very similar, it is postulated that the Drosophila rSpn27A could perhaps affect the activation of PPO in various insect systems. The hemolymph plasma from larvae of two lepidopterans (Bombyx mori and Galleria mellonella) were used and Spn27A was shown to be able to significantly block PPO activation in both species (De Gregorio, 2002b).

Even though there is a striking amino acid sequence similarity between Spn27A and PPOs in and around the cleavage site, the cleavage site itself is not identical. Specifically, Spn27A has a Lys at the cleavage site, whereas insect PPOs have an Arg at this position. Generally, trypsin-like serine proteases are known to cleave their substrates after a basic amino acid, such as Arg or Lys. The putative P1 site of rSpn27A was mutated from Lys to Ala (rSpn27A^K406A). Following this point mutation, rSpn27A^K406A completely lost its PO activation inhibitory effect. In a second set of experiments, to mimic the PPO cleavage site, the lysine of the putative P1 site was mutated to arginine (rSpn27A^K406R). Interestingly, rSpn27A^K406R inhibits PPO activation with a lower efficiency than rSpn27A wild-type in Drosophila pupal homogenate (De Gregorio, 2002b).

Serpins bind their target proteases through an RCL domain that mimics their substrate. Therefore the sequence similarity between the Spn27A RCL and the insect PPO cleavage site suggests that the target of Spn27A might be prophenoloxidase-activating enzyme (PPAE). Highly purified prophenoloxidase-activating enzyme from the coleopteran insect Holotrichia diomphalia (HdPPAE) can convert PPO to PO (Lee, 1998). Because the Drosophila PPAE has not yet been identified, highly purified PPAE from H. diomphalia was employed. To investigate whether HdPPAE is the target protease of Spn27A, a protease activity assay was conducted in the presence of rSpn27A and its mutants using chromogenic substrates. When HdPPAE and rSpn27A were incubated in a 1:1 molar ratio, the HdPPAE enzymatic activity was greatly inhibited. A similar result was obtained using the rSpn27A^K406R mutant. However, the rSpn27A^K406A mutant was unable to inhibit HdPPAE enzymatic activity (De Gregorio, 2002b).

To further demonstrate that Spn27A is a natural inhibitor of PPAE, the final step of the PPO activation system (HdPPO plus active HdPPAE with cofactors) was reconstituted in the presence of rSpn27A. The result showed that rSpn27A and the rSpn27A^K406R mutant could almost completely inhibit de novo-generated PO activity, while the rSpn27A^K406A mutant had no effect (De Gregorio, 2002b).

It is generally accepted that de novo-generated PO activity is due to limited proteolysis of PPO by the activating enzyme (PPAE). To demonstrate that the cleavage of PPO by HdPPAE is blocked by rSpn27A the effect of rSpn27A variants on the conversion of PPO to PO in the reconstituted system was checked. According to the PO activity assay it was found that rSpn27A and the rSpn27A^K406R completely inhibit the cleavage of PPO, while rSpn27A^K406A mutant has no effect. In summary, the biochemical analysis strongly suggests that Spn27A functions in Drosophila as a negative regulator of PPO activation through the inhibition of PPO-activating enzyme (PPAE) (De Gregorio, 2002b).

Expression of Spn27A is responsive to bacterial challenge

Genetic data suggest a role of Spn27A in blood-borne melanization. An antibody was developed against the C-terminal part of the protein. With this antibody, a marked protein band of ~50 kDa was detected in the hemolymph of wild-type adults. This molecular weight corresponds to that of the amino acid sequence deduced of the Spn27A gene. This 50 kDa band is absent from the hemolymph of the Spn27A^ex32 mutant flies. The band corresponding to Spn27A disappears from the hemolymph 3 h after bacterial challenge and reappears around 17 h post challenge. This disappearance is interpreted as resulting from the interaction of the serpin with its cognate protease, followed by removal or degradation of the complex. Moreover, this disappearance is infection dependent since injury with a sterile needle did not have a similar effect (Ligoxygakis, 2002b).

To determine whether the time window in which Spn27A is absent from the hemolymph correlates with increasing PO activity after infection, PO activity was examined in the hemolymph of immune-challenged wild-type flies. A rapid increase in PO activity was observed that reached its maximum at 3 h post challenge. From then on, PO activity decreased significantly following the time of serpin re-emergence in the hemolymph (17 h post challenge) and reaching basal levels 24 h following immune challenge. In parallel to each PO measurement, the hemolymph of the same infected flies was examined for the presence of the serpin. In every case, the increase in PO activity correlated with the absence of Spn27A. Conversely, reduction in PO activity coincided with Spn27A reappearance (Ligoxygakis, 2002b).

In non-infected flies deficient for Spn27A, the same elevated PO levels were observed as in wild-type flies subjected to bacterial challenge. This was even observed in some of the Spn27A mutant flies that did not show a spontaneous melanization phenotype. Finally, overexpression of Spn27A through a UAS-Spn27A construct, using the ubiquitous driver daGA4, or in the fat body of transgenic flies using yolkGAL4 inhibited the increase in PO activity normally following bacterial challenge (Ligoxygakis, 2002b).

Regulation of the melanization reaction and its activation through the Toll pathway

Depletion of Serpin-27A (Spn27A) from the hemolymph following immune challenge is best explained by assuming that it binds to a cognate protease and that the resulting complex is either removed from circulation or degraded. Two intracellular signaling pathways control the expression of challenge-induced genes in the fat body, the predominant immuno-responsive tissue of Drosophila. DNA microarray data indicate that the infection-induced expression of several putative prophenoloxidase-activating enzyme (PPAE) genes in Drosophila is under the control of the Toll pathway (De Gregorio, 2002a). Moreover, Spn27A transcription is also regulated in an immune-dependent manner (De Gregorio, 2001). It was of interest to examine whether the melanization observed in the Spn27A^ex32 mutants could be correlated to the well-defined Toll-mediated host response in Drosophila. In Dif or spaetzle (spz) mutants, in which the Toll pathway is blocked, the serpin is not removed from the circulating hemolymph. Interestingly, the serpin is removed in kenny (key) mutants that block the Imd pathway, which is not involved in the control of PPAE gene expression (De Gregorio, 2002a). A testable prediction that can be derived from these results is that in a Toll pathway mutant background PO enzymatic activity following bacterial challenge should be at very low levels. It as indeed observed that Dif- and spz-infected flies have a PO activity comparable to the basal level of non-challenged wild-type flies in contrast to key-infected flies, which show normal PO levels. The data furthermore imply that the protease (or the factor that triggers its activation), which removes Spn27A, may not be present as a zymogen activated by infection, but may need de novo protein synthesis dependent on Toll signal transduction. This was confirmed by infecting wild-type flies with bacteria in the presence of an inhibitor of translation (cycloheximide). Protein synthesis following bacterial infection was examined by mass spectrometry; none of the induced antimicrobial peptides were synthesized in the experimental conditions indicating that protein synthesis was efficiently blocked. Importantly, it was noted that in these flies during the same infection procedures the serpin was not removed from circulation. Taken together, these results on the requirement of Toll signaling pathway and protein synthesis for serpin removal show that the protease(s) removing Spn27A from circulation is synthesized de novo in response to Toll signaling. Alternatively, a component that activates it is dependent on Toll signaling-driven de novo protein synthesis (Ligoxygakis, 2002b).

The question emerging from these data is whether the above-mentioned protease or activating component is directly involved in activating melanization or whether it is required solely for Spn27A removal. In other words, does Toll signaling activate melanization directly? To address this issue, an spn27A; spz double mutant was generated. In larvae and flies homozygous for both mutations, constitutive melanization could still be observed. This means that the components of the PO cascade are already in the hemolymph, and Toll pathway activation tips the balance from an inhibitory state to protease triggering by removal of Spn27A (Ligoxygakis, 2002b).

It is concluded that Toll pathway mutants do not exhibit PO induction following bacterial challenge. In wild-type flies, PO activity reaches its maximum 3 h post-infection and decreases significantly at 17 h following immune challenge. Most of the larvae and a significant number of adults deficient for the serpin Spn27A show spontaneous melanization and increased PO activity. The results further indicate that this circulating serpin disappears from the hemolymph 3 h following bacterial challenge and re-emerges at 17 h post-infection. This time window correlates with PO activation kinetics. Moreover, overexpression of Spn27A in fat body cells of adults prevents PO activation by immune challenge. Serpin depletion (as PO activation) relies on the Toll pathway, since in mutants of this pathway Spn27A is not removed from the hemolymph. Finally, it has been shown that serpin depletion requires de novo protein synthesis, since infection in the presence of an inhibitor of translation (cycloheximide) does not result in its removal. Among the serpin-deficient flies, only those with melanotic tumors showed a constitutive expression of the antimicrobial peptide genes Drosomycin and diptericin. There is as yet no clear explanation for their expression, which is dependent on Toll and Imd respectively. Expression could be an indirect effect of the presence of the melanotic capsules in the body cavity. It is relevant to note here that other mutations, which are not specifically linked to the immune response but result in melanotic tumors, activate antimicrobial peptide gene expression in the absence of an overt microbial infection. An alternative model could be that Spn27A blocks both the Imd and Toll pathways and these pathways would be constitutively activated in flies deficient for the serpin. Although equally interesting, this possibility does not seem to be the case. If Spn27A negatively controlled these pathways, then all the serpin-deficient flies, and not only those with melanotic tumors, would exhibit expression of the peptides. Conversely, overexpression of the serpin would inhibit antimicrobial peptide induction following microbial challenge. Finally, bacterial infection of flies overexpressing the serpin (through a UAS-Spn27A transgene) results in expression of peptides at wild-type levels (Ligoxygakis, 2002b).

A surprising implication of these results is that at least one component of the melanization cascade is controlled by the Toll pathway and has to be synthesized de novo after infection. Two pieces of evidence support this hypothesis: (1) in a loss of function allele of Dif (Dif¹), which encodes a protein able to translocate to the nucleus but unable to bind DNA, depletion of serpin27A and concomitant PO activation does not take place after bacterial challenge, and (2) in the presence of protein synthesis inhibitors, Spn27A is not removed. Since the sequence found in the hinge region of serpin27A suggests that it is an active serine protease inhibitor, it is proposed that Spn27A is removed by its target protease, forming a covalent complex. Such complexes have a half-life of a few minutes and are rapidly cleared from the hemolymph, which could explain in a simple manner the observed depletion (Ligoxygakis, 2002b).

A model is presented to explain these data. In non-infection conditions, Spn27A inhibits PPAE and blocks melanization. Circulating pathogen recognition receptors sense the bacterial infection and signal to the Toll pathway. Intracellular transduction of the signal is mediated by the Rel transcription factor DIF, which initiates an acute phase transcription. This leads to de novo production of a further amount of PPAE, which induces PO cleavage and targets Spn27A for removal. Alternatively, Toll activation could lead to the production of serine protease homologs that are co-factors of PPAE, as demonstrated in a beetle (Kwon, 2000), or a modifying enzyme that triggers the cascade (like the Drosophila pipe gene). Reappearance of the serpin in the hemolymph should inhibit any further melanization-associated proteolytic action. The question whether this reappearance is a consequence of transcription was not addressed. Nevertheless, DNA microarray data show that the Spn27A transcription level is strongly elevated after septic injury (De Gregorio, 2001). Finally, the fact that in the spn27A; spz double mutant spontaneous melanization is not suppressed evokes the possibility that the Toll pathway is activating a protease dedicated to the removal of Spn27A without taking part in the actual melanization cascade. Given that Spn27A has been shown to inhibit biochemically a PPAE of another insect (De Gregorio, 2002b) and that in turn Drosophila PPAEs are rapidly upregulated following infection (De Gregorio, 2001, 2002a; Irving, 2001), the simpler scenario is favored. Analysis of an XP element insertion in a putative PPAE gene (N. Pelte and J.-M. Reichhart, unpublished data reported in Ligoxygakis, 2002) will probably help to clarify the matter (Ligoxygakis, 2002b).

Two proteases defining a melanization cascade in the immune system of Drosophila

The melanization reaction is used as an immune mechanism in arthropods to encapsulate and kill microbial pathogens. In Drosophila, the serpin Spn27A regulates melanization apparently by inhibiting the protease that activates phenoloxidase, the key enzyme in melanin synthesis. This study describes the genetic characterization of two immune inducible serine proteases, MP1 and MP2, which act in a melanization cascade regulated by Spn27A. MP1 is required to activate melanization in response to both bacterial and fungal infection, whereas MP2 is mainly involved during fungal infection. Pathogenic bacteria and fungi may therefore trigger two different melanization cascades that use MP1 as a common downstream protease to activate phenoloxidase. The melanization reaction activated by MP1 and MP2 plays an important role in augmenting the effectiveness of other immune reactions, thereby promoting resistance of Drosophila to microbial infection (Tang, 2006).

Melanization is a conserved host defense reaction in insects and other arthropods, such as in the mosquito, where it is a critical determinant of resistance to the malarial parasite. A recent key finding revealed that Spn27A, a serpin-type protease inhibitor, is a negative regulator of melanization in Drosophila. This study used suppression of the spn27A melanization phenotype as a genetic strategy to identify two Drosophila proteases, MP1 and MP2, having essential roles in activating melanization in response to microbial infection (Tang, 2006).

The data are consistent with MP2 acting genetically upstream of MP1 and thus with MP2 activating MP1 in a protease cascade leading to melanization. Interestingly, the application of evolutionary markers suggests that MP1 and MP2 belong to the primordial class of serine proteases that tend to function most downstream in a protease cascade, as in the case of Easter and its direct activator involved in activating Toll during development. However, it has not been demonstrated that MP2 directly activates MP1, since MP2 failed to cleave the zymogen form of MP1 when co-expressed in transfected Drosophila S2 cells, thereby suggesting that another protease acts in between MP2 and MP1. Since the protease down-stream of MP2 that acts either in parallel to or upstream of Spn27A, MP1 is a candidate to be PPAE, the terminal protease in the melanization cascade that cleaves prophenoloxidase (PPO) as well as the putative direct target of Spn27A. Cleavage of PPO by MP1 was not detected when assayed by co-expression in transfected S2 cells. However, biochemical studies in the beetle and tobacco hornworm have identified a non-enzymatic cofactor required for PPO cleavage by PPAE to generate PO activity in vitro (Kanost, 2004; Kown, 2000), which may indicate that such a cofactor is required to demonstrate PPO cleavage by MP1 (Tang, 2006).

The data suggest that another melanization cascade exists in Drosophila besides the one involving MP1 and MP2. In activating melanization and PO activity, MP1 is essential during both bacterial and fungal infection, whereas MP2 is essential during fungal infection and partially required during bacterial infection. Thus, another protease may function analogously to MP2 in activating MP1 during bacterial infection. The convergence of two different melanization cascades on MP1 is consistent with the idea that MP1 is the shared terminal protease of both cascades that activates phenoloxidase (Tang, 2006).

It is presumed that MP1 and MP2 activate melanization in the hemolymph. Since both MP1 and MP2 have an N-terminal signal sequence for secretion, they may be secreted by the fat body and/or blood cells into the hemolymph. This possibility is consistent with the detection of MP1 and MP2 in extracted hemolymph when epitope-tagged versions of the full-length proteases are overexpressed with act-Gal4 and with constitutive melanization induced by overexpression of preactivated MP1 using the c564-Gal4 driver, which is expressed in the fat body and hemocytes (Tang, 2006).

MP1 and MP2 define a protease cascade distinct from the one that may activate the Toll pathway; they activate melanization independently of this pathway. However, MP2 (but not MP1) is important for the induction of Drosomycin expression and for viability of Drosophila following natural fungal infection. One explanation is that MP2 activates two distinct pathways, one leading to melanization and the other leading to the induction of Drosomycin expression, and that this dual role is important for resistance to natural fungal infection. Preliminary experiments indicate that overexpression of preactivated MP2 does not induce Drosomycin expression in non-infected adult flies, unlike in the case of the Persephone protease (Ligoxygakis, 2002a), thereby suggesting that MP2 does not induce Drosomycin expression by directly activating the Toll pathway. Nonetheless, there may exist cross-talk between melanization and the Toll pathway mediated by MP2 involving an as yet undefined mechanism (Tang, 2006).

Surprisingly, the melanization reaction does not appear to be critical for survival of Drosophila after bacterial or fungal infection. However, it was observed that the inability to activate melanization is detrimental when flies are also defective in the Toll or the Imd pathway controlling antimicrobial peptide synthesis. Melanization is an immediate immune response that temporally precedes the induction of antimicrobial peptide synthesis, which requires gene transcription. Consequently, melanization may play a crucial role in weakening a microbial infection, thereby enhancing the effectiveness of subsequent immune reactions. Having a single switch, such as MP2, to activate a temporal sequence of immune reactions would therefore seem to be an advantageous mechanism for ensuring a potent defense against a microbial pathogen (Tang, 2006).

In conclusion, two serine proteases were identified, among the large set of serine proteases encoded in the Drosophila genome, as being essential components of a melanization cascade activated by microbial infection. A major goal in future studies will be to delineate the entire cascade from the pattern recognition receptor that triggers the cascade to the putative cofactor required for phenoloxidase activation (Tang, 2006).

REFERENCES

Search PubMed for articles about Drosophila serpin-27A

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date revised: 12 January 2018

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