Silvia Gigliotti

Aphidius ervi teratocytes release an extracellular enolase

We report the cloning of a gene and the characterization of the encoded protein, which is released by the teratocytes of the parasitoid Aphidius ervi in the haemocoel of the host aphid Acyrthosiphon pisum. The studied protein was identified by LC-MS/MS, and the gathered information used for isolating the full length cDNA. The corresponding gene was made of 3 exons and 2 introns, and was highly expressed in the adult wasps and in parasitized hosts. The translation product, which was named Ae-ENO, showed a very high level of sequence identity with insect enolases. In vivo immunodetection experiments evidenced Ae-ENO localization in round spots, present in the teratocytes and released in the host haemocoel. Moreover, strong immunoreactivity was detected on the surface of A. ervi larvae and of host embryos. Ae-ENO expressed in insect cells was not secreted in the medium, indicating the occurrence in the teratocytes of an unknown pathway for Ae-ENO release. The recombinant protein produced in bacteria under native conditions was a dimer, with evident enolase activity (K(m) = 0.086 +/- 0.017 mM). Enolase is a well known enzyme in cell metabolism, which, however, is associated with a multifunctional role in disease, when present in the extracellular environment, on the surface of prokaryotic and eukaryotic cells. In these cases, the enolase mediates the activation of enzymes involved in the invasion of tissues by pathogens and tumour cells, and in the evasion of host immune response. The possible role played by Ae-ENO in the host regulation process is discussed in the light of this information.

Toxoneuron nigriceps polydnavirus encodes a putative aspartyl protease highly expressed in parasitized host larvae

Toxoneuron nigriceps (Viereck) (Hymenoptera: Braconidae) is an endophagous parasitoid of larval stages of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae). This parasitoid is associated with a polydnavirus (TnBV), injected at oviposition along with the egg, and involved in the disruption of host immune reaction and endocrine balance. This paper reports the molecular characterization of TnBV2, one of the most abundant genes in the TnBV genome. TnBV2 expression produces a mature 0.6 kb transcript in fat body, prothoracic glands and haemocytes, as early as 6 h after parasitoid oviposition. Only in haemocytes a specific longer transcript of 2.5 kb is found 24 h after parasitization. The putative translation product of TnBV2 contains a retroviral type aspartyl protease domain. The possible origin and functional role of this TnBV gene are discussed.

Functional amyloids in insect immune response.

The innate immune system of insects consists of humoural and cellular responses that provide protection against invading pathogens and parasites. Defence reactions against these latter include encapsulation by immune cells and targeted melanin deposition, which is usually restricted to the surface of the foreign invader, to prevent systemic damage. Here we show that a protein produced by haemocytes of Heliothis virescens (Lepidoptera, Noctuidae) larvae, belonging to XendoU family, generates amyloid fibrils, which accumulate in large cisternae of the rough endoplasmic reticulum and are released upon immune challenge, to form a layer coating non-self objects entering the haemocoel. This amyloid layer acts as a molecular scaffold that promotes localised melanin synthesis and the adhesion of immune cells around the non-self intruder during encapsulation response. Our results demonstrate a new functional role for these protein aggregates that are commonly associated with severe human diseases. We predict that insects will offer new powerful experimental systems for studying inducible amyloidogenesis, which will likely provide fresh perspectives for its prevention.

nup154 Genetically Interacts With cup and Plays a Cell-Type-Specific Function During Drosophila melanogaster Egg-Chamber Development

Nucleoporin Nup154 is a Drosophila component of the nuclear pore complex (NPC), evolutionarily conserved from yeast to humans. While functional studies carried out in both yeast and metazoan cells indicated that Nup154 homologs are key elements of the NPC framework, the striking phenotypic specificity displayed by nup154 hypomorphic mutant alleles suggested that Nup154 might play additional roles in the context of the NPC. Actually, genetic analyses demonstrated that mutant nurse-cell nuclei do not undergo a normal chromosome dispersal process, uncovering an essential requirement for nup154 gene function during oogenesis. In this report, we show that Nup154 interacts genetically and physically with Cup, a germline-specific protein implicated in multiple aspects of female gametogenesis, including the regulation of the nurse-cell chromosome structure. The two proteins colocalize in vivo and are co-immunoprecipitated from ovarian extracts. Moreover, cup, nup154 double mutants exhibit much stronger oogenesis defects than single mutants. Our findings delineate an intriguing scenario where an ubiquitous nucleoporin might directly influence specialized developmental events.

Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism

Significance Bacillus thuringiensis and its toxins are widely used for insect control. Notwithstanding the remarkable importance of this insect pathogen, its killing mechanism has yet to be fully elucidated. Here we show that the microbiota resident in the host midgut triggers a lethal septicemia. The infection process is enhanced by reducing the host immune response and its control on replication of midgut bacteria invading the body cavity through toxin-induced epithelial lesions. The experimental approach used, leaving the midgut microbiota unaltered, allows identification of the bacterial species switching from resident symbionts to pathogens and sets the stage for developing new insect biocontrol technologies based on host immunosuppression as a strategy to enhance the impact of natural antagonists. Bacillus thuringiensis is a widely used bacterial entomopathogen producing insecticidal toxins, some of which are expressed in insect-resistant transgenic crops. Surprisingly, the killing mechanism of B. thuringiensis remains controversial. In particular, the importance of the septicemia induced by the host midgut microbiota is still debated as a result of the lack of experimental evidence obtained without drastic manipulation of the midgut and its content. Here this key issue is addressed by RNAi-mediated silencing of an immune gene in a lepidopteran host Spodoptera littoralis, leaving the midgut microbiota unaltered. The resulting cellular immunosuppression was characterized by a reduced nodulation response, which was associated with a significant enhancement of host larvae mortality triggered by B. thuringiensis and a Cry toxin. This was determined by an uncontrolled proliferation of midgut bacteria, after entering the body cavity through toxin-induced epithelial lesions. Consequently, the hemolymphatic microbiota dramatically changed upon treatment with Cry1Ca toxin, showing a remarkable predominance of Serratia and Clostridium species, which switched from asymptomatic gut symbionts to hemocoelic pathogens. These experimental results demonstrate the important contribution of host enteric flora in B. thuringiensis-killing activity and provide a sound foundation for developing new insect control strategies aimed at enhancing the impact of biocontrol agents by reducing the immunocompetence of the host.

Functional analysis of an immune gene of Spodoptera littoralis by RNAi

Insect immune defences rely on cellular and humoral responses targeting both microbial pathogens and metazoan parasites. Accumulating evidence indicates functional cross-talk between these two branches of insect immunity, but the underlying molecular mechanisms are still largely unknown. We recently described, in the tobacco budworm Heliothis virescens, the presence of amyloid fibers associated with melanogenesis in immune capsules formed by hemocytes, and identified a protein (P102) involved in their assembly. Non-self objects coated by antibodies directed against this protein escaped hemocyte encapsulation, suggesting that P102 might coordinate humoral and cellular defence responses at the surface of foreign invaders. Here we report the identification of a cDNA coding for a protein highly similar to P102 in a related Lepidoptera species, Spodoptera littoralis. Its transcript was abundant in the hemocytes and the protein accumulated in large cytoplasmic compartments, closely resembling the localization pattern of P102 in H. virescens. RNAi-mediated gene silencing provided direct evidence for the role played by this protein in the immune response. Oral delivery of dsRNA molecules directed against the gene strongly suppressed the encapsulation and melanization response, while hemocoelic injections did not result in evident phenotypic alterations. Shortly after their administration, dsRNA molecules were found in midgut cells, en route to the hemocytes where the target gene was significantly down-regulated. Taken together, our data demonstrate that P102 is a functionally conserved protein with a key role in insect immunity. Moreover, the ability to target this gene by dsRNA oral delivery may be exploited to develop novel technologies of pest control, based on immunosuppression as a strategy for enhancing the impact of natural antagonists.

The impact on microtubule network of a bracovirus IκB-like protein

Polydnavirus-encoded IκB-like proteins are similar to insect and mammalian IκB, and an immunosuppressive function in the host cells has been inferred to these proteins. Here we show that the expression of one of these IκB-like viral genes, the TnBVank1, in the Drosophila germline affects the localization of gurken, bicoid, and oskar mRNAs whose gene products are relevant for proper embryonic patterning. The altered localization of these mRNAs is suggestive of general defects in the intracellular, microtubule-based, trafficking routes. Analysis of microtubule motor proteins components such as the dynein heavy chain and the kinesin heavy chain revealed defects in the polarized microtubule network. Interestingly, the TnBVANK1 viral protein is uniformly distributed over the entire oocyte cortex, and appears to be anchored to the microtubule ends. Our data open up a very interesting issue on novel function(s) played by the ank gene family by interfering with cytoskeleton organization.

The translational repressor Cup associates with the adaptor protein Miranda and the mRNA carrier Staufen at multiple time-points during Drosophila oogenesis.

In Drosophila melanogaster, Cup acts as a translational regulator during oocyte maturation and early embryogenesis. In this report, we show that Cup associates with Miranda, an adaptor protein involved in localization of specific mRNA complexes in both neuroblasts and oocytes. miranda and cup also interact genetically, since reducing miranda activity worsens the oogenesis defects associated with different cup mutant alleles. miranda mRNA is first detected within the cytoplasm of egg chambers during early oogenesis, coincidentally with very low levels of Miranda protein. We furthermore show that Cup interacts with Staufen, a protein involved in mRNA localization during oogenesis and nervous system development, and the two proteins co-localize within the posterior cytoplasm of late oocytes. Our results substantiate the idea that Cup is a multi-functional protein cooperating with different protein partners to direct egg chamber development at multiple time-points.

The translational repressor Cup is required for germ cell development in Drosophila.

Summary In Drosophila, germ cell formation depends on inherited maternal factors localized in the posterior pole region of oocytes and early embryos, known as germ plasm. Here, we report that heterozygous cup mutant ovaries and embryos have reduced levels of Staufen (Stau), Oskar (Osk) and Vasa (Vas) proteins at the posterior pole. Moreover, we demonstrate that Cup interacts with Osk and Vas to ensure anchoring and/or maintenance of germ plasm particles at the posterior pole of oocytes and early embryos. Homozygous cup mutant embryos have a reduced number of germ cells, compared to heterozygous cup mutants, which, in turn, have fewer germ cells than wild-type embryos. In addition, we show that cup and osk interact genetically, because reducing cup copy number further decreases the total number of germ cells observed in heterozygous osk mutant embryos. Finally, we detected cup mRNA and protein within both early and late embryonic germ cells, suggesting a novel role of Cup during germ cell development in Drosophila.

Diminution of eIF4E activity suppresses parkin mutant phenotypes.

Mutations in the human parkin (PARK2) gene cause autosomal recessive-juvenile Parkinsons disease (AR-JP). In Drosophila melanogaster, mutant parkin alleles display a broad range of phenotypic alterations, including female infertility. Here we report that reducing the level of eukaryotic translation initiation factor 4E (eIF4E) activity specifically rescues the female sterile phenotypes associated with the parkin(P23) mutant allele. Additional defects, including reduction of pupal viability and body size, are also entirely recovered in both male and female flies of the abovementioned genotype. We further show that a null eIF4E-binding protein (4E-BP) allele counteracts the in vivo effects produced, in a parkin(P23) mutant background, by the reduction of functional eIF4E copy number. Moreover, Parkin and eIF4E interact in vitro and co-localize at the posterior end of developing oocytes. Finally, we show that eIF4E is over-expressed in parkin(P23) mutant ovaries as compared to wild-types. Taken together, our data are consistent with the idea that Parkin and eIF4E act in a common pathway, likely modulating cap-dependent translation initiation events.