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.

CalpA, a Drosophila calpain homolog specifically expressed in a small set of nerve, midgut, and blood cells.

Calpains are calcium-dependent proteases believed to participate in calcium-regulated signal pathways in cells. Ubiquitous calpains as well as tissue-specific calpains have been found in vertebrates. We isolated cDNA clones for a highly tissue-specific calpain gene from Drosophila melanogaster, CalpA, at 56C-D on the second chromosome. The expression of the CalpA gene product was monitored by using a specific antiserum directed against the product expressed by one cDNA clone. The encoded protein is found in a few neurons in the central nervous system, in scattered endocrine cells in the midgut, and in blood cells. In the blood cell line mbn-2, calpain is associated with a granular component in the cytoplasm. The expression of this protein is more restricted than that of the corresponding transcripts, which are widely distributed in the central nervous system, digestive tract, and other tissues. The sequence of CalpA is closely related to that of vertebrate calpains, but an additional segment is inserted in the calmodulin-like carboxy-terminal domain. This insert contains a hydrophobic region that may be involved in membrane attachment of the enzyme. Differential splicing also gives rise to a minor transcript that lacks the calmodulin-like domain.

FKBP39, a Drosophila member of a family of proteins that bind the immunosuppressive drug FK506.

A cDNA, coding for the first Drosophila melanogaster homolog of a family of proteins (FK506-binding proteins, FKBPs) which bind to the immunosuppressive drug FK506, was isolated. The deduced aa sequence corresponds to a 39-kDa product (FKBP39) which, besides a domain with similarity to FKBPs, has a highly charged domain with two strongly acidic stretches. The transcript could be detected in all developmental stages, with the highest expression in the embryo. In adult flies, the strongest signal was detected in the ovaries. Although the FKBP39 gene is expressed in the immunocompetent D. melanogaster blood cell line, mbn-2, the antibacterial defense reaction of these cells is unaffected by FK506.

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.

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.

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.

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.

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.

Partial tolerance in beta-galactosidase-transgenic mice.

A transgenic mouse line was produced which allowed the expression of E. coli beta-galactosidase (beta-Gal) under the regulatory elements of the immunoglobulin heavy chain locus. Expression of the transgene is found in spleen and bone marrow. Upon immunization of the transgenic mice with beta-Gal, a reduced but clearly detectable antibody response was obtained. Affinity purification with sera from immunized transgenic mice suggests that they contain lower affinity antibodies as compared to normal littermates. Transgenic and nontransgenic mice immunized with bovine serum albumin (BSA) alone or as a mixture with beta-Gal gave comparable anti-BSA responses. Immunization with a chemically cross-linked (Gal-BSA)-protein, however, showed a 10- to 30-fold difference in the anti-BSA response. Partial unresponsiveness to beta-Gal in the transgenic mice is best explained by a dominant, peripheral suppression mechanism linked to the antigen-presenting potential of B cells.

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.

Cloning of a VLP-protein coding gene from a parasitoid wasp Venturia canescens.

Virus-like particles (VLPs) that are bound to the egg surface of a parasitic wasp Venturia canescens are void of nucleic acid and unable to infect host tissue but instead provide a passive protection against the host's immune recognition. To investigate evolutionary and functional properties of Venturia particles, we isolated cDNAs coding for a VLP-protein by screening an expression library of the wasp, using antibodies against purified VLPs. The corresponding coding DNA is present in the wasp genome as a single-copy gene.

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.

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.

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.

Protein-specific cytotoxic T lymphocytes. Recognition of transfectants expressing intracellular, membrane-associated or secreted forms of beta-galactosidase.

BALB/c-derived tumor cells were transfected with recombinant Escherichia coli beta-galactosidase (beta-gal) genes which were inserted into IgM heavy chain gene derivatives, leading to expression of the resulting fusion protein in different cellular compartments. A beta-gal-specific, major histocompatibility complex (MHC) class I-restricted CD8+CD4- cytotoxic T lymphocyte (CTL) line of BALB/c origin raised against one transfectant expressing cytoplasmic beta-gal also lysed transfectants expressing beta-gal as membrane-inserted fusion protein, as well as transfectants secreting beta-gal. Our data show that MHC class I-restricted CTL can recognize fragments of nonviral cellular proteins, be they expressed as intracellular, membrane-inserted, or secreted products. The findings confirm and extend a hypothesis on the nature of minor histocompatibility (H) antigens formulated earlier.

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.

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.

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.

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.

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.