Proto-pyroptosis: An Ancestral Origin for Mammalian Inflammatory Cell Death Mechanism in Drosophila melanogaster.

Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. Here we provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, we unveil key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.

Possible function of two insect phospholipid-hydroperoxide glutathione peroxidases.

We compared the functional properties of two insect members of the phospholipid hydroperoxide glutathione peroxidases (PHGPx) family, VLP1, a major component of virus-like particles from the hymenopteran endoparasitoid Venturia canescens and its closest Drosophila relative, one of the putative PHGPx-proteins predicted from the Berkeley Drosophila genome sequence project. Recombinant Drosophila PHGPx shows enzymatic activity towards a number of PHGPx substrates, while the recombinant PHGPx-like domain of VLP1 lacks a functionally relevant cysteine and enzyme activity. A possible function of a non-enzymatic extracellular PHGPx-like protein is discussed.

Genetic analysis of two distinct reproductive strategies in sexual and asexual field populations of an endoparasitic wasp, Venturia canescens.

Asexual (thelytokous) females of the parasitoid Venturia canescens, which develop inside another insect, exhibit evolutionarily stable mixtures of life-history strategies, allowing two genetically distinct wasp lines to coexist sympatrically on the same host resources. Since the two asexual lines differ in a virus-like particle protein-coding gene (VLP1), the question is whether the VLP1 gene is genetically associated with the phenotype. The recent isolation of facultative sexual (arrhenotokous) and asexual V. canescens strains from the same location in Southern France has enabled an investigation of the genetic basis for the observed phenotypic differences, by comparing the two asexual lines with the corresponding homozygous VLP1 genotypes in arrhenotokous strains. This analysis showed similar patterns of morphological and functional differences exist in the ovaries of the two asexual VLP1 lines and in the two homozygous VLP1 genotypes from the field, suggesting that the VLP1 gene alteration either causes the ovarian phenotype or is genetically closely linked to the putative gene. However, the VLP1-gene may not be the only gene contributing to the phenotypic effects observed in the asexual lines. Although the two VLP1-alleles segregate with the relative differences in the ovary distribution of eggs, the absolute egg numbers differ in the corresponding asexual and sexual genotypes. This suggests that an additional unlinked gene may be involved in the transfer of eggs from the ovarioles into the oviduct.

Insect hemolymph clotting: evidence for interaction between the coagulation system and the prophenoloxidase activating cascade.

Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the alpha-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.

The coagulation of insect hemolymph.

In contrast to both vertebrates and non-insect arthropods, little is known about the coagulation of hemolymph (hemostasis) in insects. We discuss the integration of the hemostatic response with other branches of the insect immune system. We also describe the present stage in the characterization of both soluble and cellular factors that contribute to hemostasis in insects. The factors of the well-characterized clotting cascades of vertebrates, primitive chelicerates and crustaceans are used to assess the implications of sequencing the whole Drosophila genome for searching candidate genes involved in hemostasis. Some striking similarities between blood clotting in vertebrates and the reaction of insect cells involved in hemolymph coagulation have implications for a phylogenetic comparison of hemostasis between divergent animal classes.

Innate immunity and its evasion and suppression by hymenopteran endoparasitoids.

Recent studies suggest that insects use pattern recognition molecules to distinguish prokaryotic pathogens and fungi from "self" structures. Less understood is how the innate immune system of insects recognizes endoparasitic Hymenoptera and other eukaryotic invaders as foreign. Here we discuss candidate recognition factors and the strategies used by parasitoids to overcome host defense responses. We suggest that host-parasitoid systems are important experimental models for studying how the innate immune system of insects recognizes foreign invaders that are phylogenetically more closely related to their hosts. The strategies used by parasitoids suggest that insects may employ "hidden-self" recognition molecules for attacking foreign objects intruding the open circulatory system. BioEssays 23:344-351, 2001.

Changes in glycosylation during Drosophila development. The influence of ecdysone on hemomucin isoforms.

To explore a possible signal function of glycodeterminants and the tissue specificity of glycosylation in Drosophila melanogaster, hemomucin, a surface mucin previously isolated from cell lines was studied. It was shown to exist in two glycoforms with molecular masses of 100 and 105 kDa, respectively. The two forms differ by the presence of O-linked galactose, which was only detected in the larger glycoform using the beta-galactose specific peanut agglutinin (PNA). The 105 form was found in cell lines after addition of the cell cycle inhibitor taxol and after induction with ecdysone. When whole animal tissues were analyzed using PNA, dramatic changes were observed during development. We were able to identify a number of proteins, which showed strong PNA-staining in stages with a high ecdysone titer, while virtually no staining was detected in adults. This pattern was specific for PNA and was not observed with any of the other lectins employed in this study. Surprisingly, in contrast to our observation in cell lines, PNA staining of hemomucin was not observed in late third larval and pupal stages, which are known to produce high ecdysone titers. The only organ, in which significant amounts of the 105 form were detected, were the ovaries, where hemomucin is produced in follicle cells during the late phase of oogenesis and subsequently incorporated into the chorion.

Two distinct reproductive strategies are correlated with an ovarian phenotype in co-existing parthenogenetic strains of a parasitic wasp.

The question whether different organisms are able to compete for the same resource is of fundamental importance to evolutionary biology. Sympatric co-existence of similar species on a single resource has long been claimed to be unstable. However, indirect evidence suggests that parasitic wasps exhibit evolutionarily stable mixtures of life-history strategies. Here we describe genetically distinct strains of a parthenogenetic wasp Venturia canescens, with different ovarian phenotypes that affect egg numbers in oviducts. Wasp females with large egg load search for caterpillars and deposit eggs immediately after host encounter, whereas females with fewer eggs delay parasitism. Since the outcome of interlarval competition within super-parasitized caterpillars depends on the age distribution of competing larvae, the two egg deposition strategies may co-exist under conditions that favor super-parasitism.

Evidence for serine protease inhibitor activity in the ovarian calyx fluid of the endoparasitoid Venturia canescens.

Endoparasitic wasps are able to develop inside permissive host insects due to their ability to overcome or evade the host's immune system. In the present study, we provide experimental evidence that ovarian calyx fluid of the ichneumonid endoparasitoid Venturia canescens has the potential to alter host haemocyte spreading and inhibit host haemolymph melanisation due to the presence of a putative serine protease inhibitor (serpin) activity. The existance of a serpin-like activity in the calyx fluid is also supported by experiments where the synthetic protease inhibitor p-APMSF had effects on cellular and cell-free immune reactions similar to ovarian calyx fluid. In addition, based on proteolytic digestion patterns of a wasp egg surface protein, we predict an Arg-specific trypsin-like protease activity in the host haemolymph which is possibly affected by calyx fluid components as well. Our data suggest that ovarian calyx fluid, deposited into the host together with the parasitoid egg, contains serpin activity which might transiently inactivate host defence reactions until other means of protection are established on the egg surface.

Animal and plant members of a gene family with similarity to alkaloid-synthesizing enzymes.

Here we describe novel members of a gene family which have similarity to strictosidine synthase (SS), one of the key enzymes in the production of monoterpene indole alkaloids. In addition to the first animal member of the family described previously (Drosophila hemomucin), a second Drosophila member has been identified, which appears to differ in subcellular distribution from hemomucin. In Arabidopsis, SS-like genes form a multigene family, compatible with a possible function as antifeedants and antibacterial compounds. In Caenorhabditis, two members have been identified and one member each in mouse and human. Interestingly, the human SS-like gene is strongly expressed in the brain, the very organ many of the indole alkaloids act upon.

Insect glycobiology: a lectin multigene family in Drosophila melanogaster.

Glycodeterminants play an important role in mediating cellular and cell-substrate interactions during development and immune-related reactions enabling an organism to distinguish self determinants from non-self or modified-self determinants. One of the hallmarks of sugar recognition molecules (lectins) is their wide range of binding activities and their organisation in multigene families. Here we describe a group of Drosophila genes that are possible members of the C-type lectin family.

A maternal gene mutation correlates with an ovary phenotype in a parthenogenetic wasp population.

Endoparasitoid wasps rely on maternal protein secretions, including viruses and virus-like particles (VLPs), to overcome host defense reactions. In the ichneumonid Venturia canescens, VLPs are assembled in the nuclei of ovarian calyx gland cells, secreted into the lumen of the gland, and eventually transmitted into the host caterpillar together with the parasitoid egg. One of the genes coding for VLP proteins, termed VLP1, exists in two alleles producing two structurally different proteins. Here we describe the establishment and initial phenotypic characterisation of two parthenogenetic laboratory strains, which differ in VLP1 as well as in other genetic markers. A comparison of calyx tissues from the two strains revealed morphological differences that seem to affect egg movement from the ovarioles into the oviduct. The observed histological changes are correlated with differences in egg maturation and embryonic development causing a delay in larval hatching in one of the strains. Under conditions that favour superparasitism, the two strains differ in the number of offspring produced.

Is the surface of endoparasitic wasp eggs and larvae covered by a limited coagulation reaction?

Evidence is presented for the existence of a Venturia homologue of hemomucin, a surface mucin that was recently described in Drosophila. Venturia hemomucin is part of the mucinous layer on the egg and larval surface of the parasitoid. Venturia hemomucin forms a complex with lipophorin and other host hemolymph components, that is similar to a hemomucin-lipophorin complex which is part of the coagulation reaction. The possible formation of a specific layer against the host defence system is discussed for eggs and larvae that develop inside another insect.

TER94, a Drosophila homolog of the membrane fusion protein CDC48/p97, is accumulated in nonproliferating cells: in the reproductive organs and in the brain of the imago.

We have cloned a Drosophila homolog of the membrane fusion protein CDC48/p97. The open reading frame of the Drosophila homolog encodes an 801 amino acid long protein (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in imago, but is suppressed in larva. In the imago, the immunoreactivity was exclusively present in the head and in the gonads of both sexes. In the head the most striking staining was observed in the entire neuropil of the mushroom body and in the antennal glomeruli. Besides TER94, sex-specific forms were also detected in the gonads of the imago: p47 in the ovaries and p98 in the testis. TER94/p47 staining was observed in the nurse cells and often in the oöcytes, while TER94/p98 staining was present in the sperm bundles. On the basis of its distribution we suggest that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles.

A protein with protective properties against the cellular defense reactions in insects.

The molecular mechanism of how insects recognize intruding microorganisms and parasites and distinguish them from own body structures is not well known. We explored evolutionary adaptations in an insect parasitoid host interaction to identify components that interfere with the recognition of foreign objects and cellular encapsulation. Because some parasitoids provide protection for the developing wasp in the absence of an overt suppression of the insect host defense, we analyzed the surface of eggs and symbiotic viruses for protective properties. Here we report on the molecular cloning of a 32-kDa protein (Crp32) that is one of the major protective components. It is produced in the calyx cells of the female wasp ovaries and attached to the surface of the egg and other particles including polydnaviruses. The recombinant protein confers protection to coated objects in a cellular encapsulation assay suggesting that a layer of Crp32 may prevent cellular encapsulation reactions by a local inactivation of the host defense system.

A polydnavirus-encoded protein of an endoparasitoid wasp is an immune suppressor.

The molecular mechanism by which polydnaviruses of endoparasitoid wasps disrupt cell-mediated encapsulation reactions of host insects is largely unknown. Here we show that a polydnavirus-encoded protein, produced from baculovirus and plasmid expression vectors, prevents cell surface exposure of lectin-binding sites and microparticle formation during immune stimulation of haemocytes. The inactivation of immune-related cellular processes by this protein was analysed using a specific lectin and annexin V and shown to be virtually identical to polydnavirus-mediated effects on haemocytes. Cytochalasin D application has similar effects on haemocytes, suggesting that the immune suppression by the polydnavirus protein is caused by the destabilization of actin filaments. Since the exposure of cell surface glycoproteins and the formation of microparticles are part of an immune response to foreign objects or microorganisms and a prerequisite for cell-mediated encapsulation of microorganisms and parasites, the virus-encoded protein may become an important tool for the inactivation of cellular immune reactions in insects and an essential component in understanding immune suppression in parasitized host insects.

Helix pomatia lectin and annexin V, two molecular probes for insect microparticles: possible involvement in hemolymph coagulation.

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail (Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.

Multiple alleles encoding a virus-like particle protein in the ichneumonid endoparasitoid Venturia canescens.

Hymenopteran endoparasitoids produce nuclear secretions from ovarian glands, which are deposited into the host insect together with the egg, protecting the developing parasitoid against the host's defence reactions. In the ichneumonid Venturia canescens, virus-like particles (VLPs), are attached to the egg surface and provide passive protection against encapsulation by the host. One of the four major particle proteins (p40) is expressed not only in the calyx gland but also in tissues that are not involved in particle production. The p40 coding DNA from V. canescens was cloned and sequenced. Within the coding DNA a tandem repeat sequence, coding for a putative proteolytic cleavage site of the PEST type, is rearranged in a significant portion of the wasp population. A corresponding polymorphism was also detected in the protein. The amino-terminal region of the deduced protein contains a putative type II transmembrane domain. The carboxy-terminal region shows similarity to the phospholipid hydroxyperoxide glutathione peroxidase (PHGPX) of vertebrates. A peroxidase function of the p40, although not ruled out, is unlikely due to the absence of a reactive centre which is typical for many vertebrate peroxidases. The overall conservation of the hydropathic region is discussed in the context of the formation of the viral envelope and its possible function in the immune protection.

Helix pomatia lectin, an inducer of Drosophila immune response, binds to hemomucin, a novel surface mucin.

We describe the isolation and initial characterization of hemomucin, a novel Drosophila surface mucin that is likely to be involved in the induction of antibacterial effector molecules after binding a snail lectin (Helix pomatia A hemagglutinin). Two proteins of 100 and 220 kDa were purified from the membrane fraction of a Drosophila blood cell line using lectin columns. The two proteins are products of the same gene, as demonstrated by peptide sequencing. The corresponding cDNAs code for a product that contains an amino-terminal putative transmembrane domain, a domain related to the plant enzyme strictosidine synthase, and a mucin-like domain in the carboxyl-terminal part of the protein. The gene is expressed throughout development. In adult flies, high expression is found in hemocytes, in specialized regions of the gut, and in the ovary, where the protein is deposited onto the egg surface. In the gut, the mucin co-localizes with the peritrophic membrane. The cytogenetic location of the gene is on the third chromosome in the region 97F-98A.

HLH106, a Drosophila transcription factor with similarity to the vertebrate sterol responsive element binding protein.

We cloned a Drosophila homolog to the sterol responsive element binding proteins (SREBPs). In vertebrates, the SREBPs are regulated by a mechanism that involves cleavage of the protein that normally residues in the cellular membranes and translocation of the released transcription factor into the nucleus. Regulation of the Drosophila factor HLH106 apparently follows the same mechanism, and we find the full-length gene product in the membrane fraction and a shorter cross-reacting form in the nuclear fraction. This nuclear form, which may correspond to proteolytically activated HLH106, is abundant in the blood cell line mbn-2. The general domain structure of HLH106 is very similar to that in SREBP. HLH106 is expressed throughout development, and it is present at high levels in Drosophila cell lines. In contrast to the rat homolog, HLH106 transcripts are not more abundant in adipose tissue than in other tissues.