InteractiveFly: GeneBrief

Serpin77Ba: Biological Overview | References

Epithelial tissues facing the external environment are essential to combating microbial infection. In addition to providing a physical barrier, epithelial tissues mount chemical defenses to prevent invasion of internal tissues by pathogens. The melanization reaction implicated in host defense is activated in the respiratory system, the trachea, of Drosophila. Tracheal melanization can be activated by the presence of microorganisms but is normally blocked by Spn77Ba, a protease inhibitor in the serpin family. Spn77Ba inhibits a protease cascade involving the MP1 and MP2 proteases that activates phenol oxidase, a key enzyme in melanin biosynthesis. Unexpectedly, it was found that tracheal melanization resulting from Spn77Ba disruption induces systemic expression of the antifungal peptide Drosomycin via the Toll pathway. Such signaling between local and systemic immune responses could represent an alarm mechanism that prepares the host in case a pathogen breaches epithelial defenses to invade internal tissues (Tang, 2008).

Innate immunity is an ancient system of host defense against pathogens used by a wide range of organisms. In humans, innate immunity is the front line of host defense that acts before the adaptive immune system. Whereas the adaptive immune system can be programmed to recognize an almost infinite number of specific antigens, the innate immune system is hardwired to recognize a limited number of common determinants found in infectious agents. Nonetheless, the innate immune system involves a sophisticated repertoire of humoral and cellular responses, acting at local as well as systemic levels, which together provide an effective barrier to infection by pathogens. In recent years, many fundamental mechanisms and principles of innate immunity have been revealed from studies with the fruit fly Drosophila (Tang, 2008).

A key innate immune mechanism found in both humans and Drosophila involves signaling by receptors in the Toll family. In humans, upon microbial infection, Toll-like receptors (TLRs) activate the synthesis of cytokines as well as other molecules that stimulate induction of the adaptive immune system. In Drosophila, Toll activates the synthesis of antimicrobial peptides (AMPs) by the fat body, the functional equivalent of human liver and adipose tissue. Since AMPs synthesized by the fat body are secreted into the blood-like hemolymph which bathe all internal tissues and organs, they provide systemic protection against pathogens that may invade the body cavity. In humans, pathogens are directly sensed by TLRs, but in Drosophila, they are detected by circulating pattern recognition receptors (PRRs) that trigger protease cascades, which lead to cleavage of the Spätzle protein to generate the ligand that activates Toll signaling. In Drosophila, the Toll pathway is mainly responsive to infection by Gram-positive bacteria and fungi, whereas the Imd pathway activates AMP synthesis in response to infection by Gram-negative bacteria. Both pathways, as well as human TLRs, trigger immune gene expression by activating a transcription factor in the NF-κB family (Tang, 2008).

Another major immune response in Drosophila, and more generally in insects and other arthropods, is the melanization reaction (Cerenius, 2004). Melanization involves the rapid synthesis of melanin at the site of infection and injury in order to contain a microbial pathogen as well as to facilitate wound healing. A key enzyme in melanin biosynthesis is phenol oxidase (PO), which catalyzes the oxidation of phenols to quinones that then polymerize into melanin. A by-product of PO activity is reactive oxygen species (ROS), a potent antimicrobial agent. PO is synthesized as an inactive zymogen called proPO (PPO), which is released by crystal cells that circulate in the hemolymph and cleaved to generate active PO at the end of a protease cascade (Cerenius, 2004). The proteases in this cascade also exist as inactive zymogens in the hemolymph, analogous to components of the complement pathway found in human blood and tissues. Thus, like the human complement pathway, the melanization reaction functions as a component of the humoral immune response to contain pathogens that invade internal body spaces (Tang, 2008).

As illustrated by the Toll pathway and the melanization reaction in Drosophila but also by the complement pathway in humans, a recurring theme in innate immunity is the use of protease cascades to activate immune responses. Such protease cascades, because of their potential for enormous signal amplification, are advantageous for detecting minute levels of pathogens. However, tight regulation must exist to prevent their activation under normal conditions and to localize their action both temporally and spatially. Indeed, C1 inhibitor, a member of the serpin family of protease inhibitors, is an essential regulator of the complement pathway (Wagenaar-Bos, 2006). Moreover, genetic studies in Drosophila have defined important roles for serpins in inhibiting the protease cascades that activate Toll and the melanization reaction (De Gregorio, 2002; Levashina, 1999; Ligoxygakis, 2002b; Tang, 2008 and refereces therein).

Epithelial tissues exposed to microorganisms in the environment play an important role in innate immunity. In addition to providing a physical barrier, such epithelia secrete chemical defenses to prevent pathogen penetration of internal tissues. Recent studies have shown that epithelia in Drosophila, as in humans, mount local immune responses. In particular, the respiratory and reproductive tracts express the antifungal peptide Drosomycin (Drs) in response to contact with pathogens, but interestingly, this response depends on the Imd pathway, not the Toll pathway utilized by the fat body during systemic antimicrobial responses. Moreover, reactive oxygen species generated by a dual oxidase are essential for the antimicrobial activity of the gastrointestinal tract (Ha, 2005; Tang, 2008 and references therein).

The melanization reaction can be activated in the trachea, the respiratory system, of Drosophila. In an RNAi screen to investigate the function of serpins in Drosophila, it was discovered that the serpin Spn77Ba is a trachea-specific inhibitor of melanization. As in other tissues, melanization of the trachea is strongly enhanced by the presence of microorganisms. Surprisingly, however, tracheal melanization induces a systemic response (Drs expression in the fat body) via the Toll pathway, which may represent a mechanism for alerting and preparing the host for possible infection of internal tissues. These data suggest that melanization is an intrinsic immune response of respiratory epithelium under tight negative control by a serpin (Tang, 2008).

Transgenic flies were generated bearing an inverted repeat construct of serpin Spn77Ba under control of the UAS element recognized by the Gal4 transcriptional activator (UAS-Spn77Ba-IR). These flies were crossed to Gal4 'driver' flies in order to activate RNAi of Spn77Ba in the progeny. When the act-Gal4 driver was used to activate Spn77Ba RNAi in virtually all tissues during development, the result was a striking phenotype in larva: melanization of the tracheal system, the respiratory organ. Tracheal melanization was first visible during the second or third instar larval stage and increased in intensity until the affected larva died within a few days before reaching the pupal stage. This phenotype was observed in experiments with two different inverted repeat constructs targeting different regions of the Spn77Ba gene and with multiple independently generated transgenic lines for each construct. By quantitative RT-PCR analysis, it was estimated that RNAi was able to knock down Spn77Ba expression to 20%-30% of the wild-type level. These results suggest that Spn77Ba is required for inhibiting melanization in the tracheal system and for larval viability (Tang, 2008).

A spn77Ba null mutation, l(3)77ABi[W7], was identified that causes early lethality, which was rescued by overexpression of the Spn77Ba protein using the act-Gal4 driver. To bypass this early essential requirement for Spn77Ba function, Spn77Ba was overexpressed in the l(3)77ABi[W7] mutant background, using the hs-Gal4 driver. At 18°C, leaky expression of Spn77Ba was sufficient for some larvae to grow to the second instar stage, and these survivors had melanized trachea and in most cases died before reaching the third instar stage. At 22°C-25°C, some larvae survived to third instar and even grew to pupae. These larvae and pupae also had melanized trachea, which was similar to that induced by Spn77Ba RNAi. These results confirm that l(3)77ABi[W7] is a mutation of the spn77Ba gene and that the phenotype induced by RNAi is due to specific knockdown of the spn77Ba gene. They also suggest that low level of Spn77Ba activity, such as left by RNAi, was sufficient to fulfill Spn77Ba's essential function during early development, but not its role in inhibiting tracheal melanization during the larval stage (Tang, 2008).

Spn77Ba regulates tracheal melanization by inhibiting a protease cascade that activates phenol oxidase, a key enzyme in melanin biosynthesis. This protease cascade involves the proteases MP1 and MP2, which previously were shown to regulate the melanization reaction in the hemolymph where it is inhibited by the serpin Spn27A (Tang, 2006). While hemolymph melanization involves MP1 and MP2 made by fat body cells and/or hemocytes, tracheal melanization involves the same proteases locally synthesized by tracheal cells. Spn77Ba is required, as well as expressed, in tracheal cells to prevent tracheal melanization. A previous study showed that the cuticular PO involved in host defense and wound healing in the silkworm is transported from the hemolymph (Ashida, 1995). In contrast, the current results obtained with the Bc¹ mutation indicate that tracheal melanization is not mediated by hemolymph PO. In preliminary experiments, tracheal-specific RNAi of each of several PO genes (CG2952, CG5779, CG8193, CG10484) did not suppress tracheal melanization resulting from Spn77Ba RNAi, perhaps indicating that multiple PO genes are responsible for tracheal melanization. Interestingly, the Rel^E20 mutation in Relish, a transcription factor in the Imd pathway, partially suppressed tracheal melanization and overall Drs expression resulting from Spn77Ba RNAi, which suggests that Rel could be important for the expression of a component required for tracheal melanization (Tang, 2008).

Tracheal melanization is inducible by microorganisms. Thus, tracheal melanization may be a local immune response analogous to the local synthesis of reactive oxygen species and antimicrobial peptides by epithelia facing the external environment. Such a role is consistent with the observation that Spn77Ba is localized in the extracellular space facing the tracheal lumen. Melanization could be used to encapsulate pathogens killed by these other local immune responses, and reactive oxygen species generated during the melanization reaction may aid in killing pathogens as well. Melanization could also create a physical barrier to impede pathogen penetration of the tracheal epithelium, as in the case where melanin is deposited at the site of entry in defense against entomopathogenic fungi that infect by crossing the insect cuticle (Vey, 1986). Spn77Ba overexpression or MP1 RNAi did not make larvae more susceptible to natural B. bassiana infection. However, testing of more pathogens will be needed to determine the importance of melanization in combating infection (Tang, 2008).

An issue that remains to be addressed is the mechanism by which microorganisms activate the melanization cascade in the tracheal system. Overexpression of PGRP-LE, a pattern recognition receptor (PRR) expressed in tracheal epithelia, does not lead to constitutive tracheal melanization (Takehana, 2002; Takehana, 2004), suggesting that this PRR is not a tracheal-specific activator of melanization. Interestingly, under axenic conditions, Spn77Ba RNAi flies still showed tracheal melanization, although at a lower strength than seen under normal conditions or in the presence of bacteria. This low level of melanization could reflect activation of the melanization cascade by dead microorganisms present in the culture food medium. Alternatively, even in the absence of microbial infection, the target protease of Spn77Ba could exist in the tracheal system already activated rather than entirely as inactive zymogen, but is prevented from activating the whole cascade and thus melanization by the presence of Spn77Ba. In this case, microbial infection could trigger tracheal melanization by either reducing the level of Spn77Ba or increasing the amount of activated target protease. Defining the actual mechanism may require identifying a natural pathogen that strongly induces tracheal melanization (Tang, 2008).

Regardless of how it is activated, tracheal melanization appears to play a key intermediary role in inducing systemic expression of the antifungal peptide gene Drosomycin (Drs). It had previously been reported that melanotic 'tumors' resulting from melanization of aberrant host tissue are associated with systemic Drs expression, but the mechanism linking melanization and Drs expression has been obscure (Ligoxygakis, 2002b; Scherfer, 2006). The data suggest that melanization induces Drs expression, rather than the two immune responses being triggered in parallel, and that a product of the melanization reaction is involved in inducing Drs expression. However, as melanization does not necessarily activate Drs expression in all other contexts, this product may not be melanin itself but rather a melanin metabolite or a secondary signal induced when melanization is activated in the trachea. Indeed, the mechanical and ROS-related insults resulting from tracheal melanization may themselves induce a stress response that contributes to the systemic induction of Drs. In any case, it appears that hemocytes are not involved in transmitting signals that connect tracheal melanization to fat body Drs expression, since l(3)hem larvae lacking hemocytes retain the systemic response (Tang, 2008).

Whatever the molecular nature of this product, it is presumably a diffusible molecule that can pass through the tracheal system basement membrane into the hemolymph, where it activates the Toll pathway to induce Drs synthesis by the fat body. Moreover, it apparently acts upstream or at the level of the Persephone (Psh) protease involved in cleaving Spätzle to generate the Toll ligand. Interestingly, it was recently shown that Psh, which specifically functions to activate Toll in response to fungal infection, is proteolytically activated by a secreted fungal protease and thus acts as a direct sensor of this virulence factor (Gottar, 2006). These data suggest that local melanization is involved in inducing Drs expression after fungal infection and that Psh can be activated by an alternative mechanism in which a host factor arising from melanization triggers Psh activation, which could be analogous to a danger signal from damaged tissue that activates the immune system. It is therefore speculated that induction of Drs synthesis by tracheal melanization represents signaling between local and systemic immune responses that alerts and prepares the host for potential invasion of internal tissues by pathogens such as entomopathogenic fungi. Such an alarm system could be advantageous for organisms in which pathogens are naturally first encountered at epithelial surfaces (Tang, 2008).

Search PubMed for articles about Drosophila Spn77Ba

Ashida, M. and Brey, P. T. (1995). Role of the integument in insect defense: pro-phenol oxidase cascade in the cuticular matrix. Proc. Natl. Acad. Sci. 92: 10698-10702. PubMed ID: 11607587

Cerenius, L. and Soderhall, K. (2004). The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198: 116-126. PubMed ID: 15199959

De Gregorio, E., et al. (2002). An immune-responsive Serpin regulates the melanization cascade in Drosophila. Dev. Cell 3: 581-592. PubMed ID: 12408809

Gottar, M., et al. (2006). Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127: 1425-1437. PubMed ID: 17190605

Ha, E. M., et al. (2005). A direct role for dual oxidase in Drosophila gut immunity. Science 310: 847-850. PubMed ID: 16272120

Levashina, E. A., et al. (1999). Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila. Science 285: 1917-1919. PubMed ID: 10489372

Ligoxygakis, P., et al. (2002a). Activation of Drosophila Toll during fungal infection by a blood serine protease. Science 297: 114-116. PubMed ID: 12098703

Ligoxygakis, P., et al. (2002b). A serpin mutant links Toll activation to melanization in the host defence of Drosophila. EMBO J. 21: 6330-6337. PubMed ID: 12456640

Scherfer, C., et al. (2006). The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation. Dev. Biol. 295: 156-163. PubMed ID: 16690050

Takehana, A., et al. (2002). Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae. Proc. Natl. Acad. Sci. 99: 13705-13710. PubMed ID: 12359879

Takehana, A., et al. (2004). Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity. EMBO J. 23: 4690-4700. PubMed ID: 15538387

Tang, H., Kambris, Z., Lemaitre, B. and Hashimoto, C. (2006). Two proteases defining a melanization cascade in the immune system of Drosophila, J. Biol. Chem. 281: 28097-28104. PubMed ID: 16861233

Tang, H., Kambris, Z., Lemaitre, B. and Hashimoto, C. (2008). A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev. Cell 15(4): 617-26. PubMed ID: 18854145

Vey, A. and Gotz, P. (1986). Antifungal cellular defense mechanisms in insects. In: Hemocytic and Humoral Immunity in Arthropods, A.P. Gupta, editor. John Wiley and Sons Inc, New York 89-115.

Wagenaar-Bos, I. G. and Hack, C. E. (2006) Structure and function of C1-inhibitor. Immunol. Allergy Clin. North Am. 26: 615-632. PubMed ID: 17085281

date revised: 10 June 2009

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.