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 (rSpn27AK406A). Following this point mutation, rSpn27AK406A 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 (rSpn27AK406R). Interestingly, rSpn27AK406R 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 rSpn27AK406R mutant. However, the rSpn27AK406A 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 rSpn27AK406R mutant could almost completely inhibit de novo-generated PO activity, while the rSpn27AK406A 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 rSpn27AK406R completely inhibit the cleavage of PPO, while rSpn27AK406A 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).
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 Spn27Aex32 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).
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 Spn27Aex32 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).
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).
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