Lack of an antibacterial response defect in Drosophila Toll-9 mutant

Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.     

Signaling mechanisms in the antimicrobial host defense of Drosophila

Drosophila has appeared in recent years as a powerful model to study innate immunity. Several papers published in the past year shed light on the role of the three Rel proteins Dorsal, Dif and Relish in the regulation of antimicrobial peptide expression. In addition, the discovery that a blood serine protease inhibitor is involved in the control of the antifungal response indicates that Toll is activated upon triggering of a proteolytic cascade and does not function as a Drosophila pattern recognition receptor.     

Plant defense and antimicrobial peptides

Plants are constantly exposed to a large array of pathogenic organisms and the survival in these conditions demands quick defense responses which include the synthesis of defense peptides and proteins with antimicrobial properties. The main groups of antimicrobial peptides found in plants are thionins, defensins and lipid transfer proteins. They constitute interesting candidates to engineer disease resistance in plants.     

Superoxide activates constitutive nitric oxide synthase in a brain particulate fraction

Nitric oxide (*NO) can act as an antioxidant by directly scavenging reactive free radicals, inhibiting the oxidative chemistry of iron, and signaling the up-regulation of antioxidant enzymes. However, the cellular utility of *NO as an antioxidant requires that constitutive nitric oxide synthase (NOS) be activated rapidly by a signal(s) for oxidant formation. We report here that superoxide (O2*-), added directly as potassium superoxide (KO2), produced a superoxide dismutase-sensitive and hydrogen peroxide-independent stimulation of NOS activity, measured by the conversion of [3H]arginine to [3H]citrulline and nitrite formation, in a synaptic particulate fraction from rat brain cerebral cortex. O2*- produced maximal activation of NOS in the presence of the antioxidant urate and ATP. Stimulation of NOS activity by O2*- was abolished by N-monomethyl-L-arginine and by the Ca2+ chelator EGTA but not by 7-nitroindazole, which would be expected to inhibit neuronal NOS. We propose that limited activation of NOS by O2*- may be an important contributor to brain oxidant defenses and, more generally, a signal for cellular adaptation and survival, although excessive generation of nitrogen oxides would be expected to produce neurotoxicity.     

The apoptosome activates caspase-9 by dimerization

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.     

High herbivore pressure favors constitutive over induced defense

Theoretical and empirical studies show that, when past or current herbivory is a reliable cue of future attack and defenses are costly, defenses can be induced only when needed and thereby permit investment in other functions such as growth or reproduction. Theory also states that, in environments where herbivory is constantly high, constitutive defenses should be favored. Here, we present data to support the second aspect of the induced resistance hypothesis. We examined herbivore‐induced responses for four species of Inga (Fabaceae), a common canopy tree in Neotropical forests. We quantified chemical defenses of expanding leaves, including phenolic, saponin and toxic amino acids, in experimental field treatments with and without caterpillars. Because young leaves lack fiber and are higher in protein than mature leaves, they typically lose >25% of their leaf area during the few weeks of expansion. We predicted that the high rates of attack would select for investment in constitutive defenses over induction. Our data show that chemical defenses were quite unresponsive to herbivory. We demonstrated that expanding leaves showed no or only small increases in investment in secondary metabolites, and no qualitative changes in the phenolic compound profile in response to herbivory. The proteinogenic amino acid tyrosine, which can be toxic at high concentrations, showed the greatest levels of induction. Synthesis: These results provide some of the first support for theoretical predictions that the evolution of induced vs. constitutive defenses depends on the risk of herbivory. In habitats with constant and high potential losses to herbivores, such as tropical rainforests, high investments in constitutive defenses are favored over induction.     

Induction of neuron-specific glycosylation by Tollo/Toll-8, a Drosophila Toll-like receptor expressed in non-neural cells

Specific glycan expression is an essential characteristic of developing tissues. Our molecular characterization of a mutation that abolishes neural-specific glycosylation in the Drosophila embryo demonstrates that cellular interactions influence glycan expression. The HRP epitope is an N-linked oligosaccharide expressed on a subset of neuronal glycoproteins. Embryos homozygous for the TM3 balancer chromosome lack neural HRP-epitope expression. Genetic and molecular mapping of the relevant locus reveals that Tollo/Toll-8, a member of the Toll-like receptor family, is altered on the TM3 chromosome. In wild-type embryos, Tollo/Toll-8 is expressed by ectodermal cells that surround differentiating neurons and precedes HRP-epitope appearance. Re-introduction of Tollo/Toll-8 into null embryos rescues neural-specific glycan expression. Thus, loss of an ectodermal cell surface protein alters glycosylation in juxtaposed differentiating neurons. The portfolio of expressed oligosaccharides in a cell reflects its identity and also influences its interactions with other cells and with pathogens. Therefore, the ability to induce specific glycan expression complements the previously identified developmental and innate immune functions of Toll-like receptors.     

Balancing STING in antimicrobial defense and autoinflammation

Rapid detection of microbes is crucial for eliciting an effective immune response. Innate immune receptors survey the intracellular and extracellular environment for signs of a microbial infection. When they detect a pathogen-associated molecular pattern (PAMP), such as viral DNA, they alarm the cell about the ongoing infection. The central signaling hub in sensing of viral DNA is the stimulator of interferon genes (STING). Upon activation, STING induces downstream signaling events that ultimately result in the production of type I interferons (IFN I), important cytokines in antimicrobial defense, in particular towards viruses. In this review, we describe the molecular features of STING, including its upstream sensors and ligands, its sequence and structural conservation, common polymorphisms, and its localization. We further highlight how STING activation requires a careful balance: its activity is essential for antiviral defense, but unwanted activation through mutations or accidental recognition of self-derived DNA causes autoinflammatory diseases. Several mechanisms, such as post-translational modifications, ensure this balance by fine-tuning STING activation. Finally, we discuss how viruses evade detection of their genomes by either exploiting cells that lack a functional DNA sensing pathway as a niche or by interfering with STING activation through viral evasion molecules. Insight into STING’s exact mechanisms in health and disease will guide the development of novel clinical interventions for microbial infections, autoinflammatory diseases, and beyond.     

Involvement of mytilins in mussel antimicrobial defense

Four cationic peptides were purified from mussel (Mytilus galloprovincialis) hemocytes. A combination of Edman degradation and mass spectrometry of plasma revealed (i) a previously characterized molecule, mytilin B (Charlet, M., Chernysh, S., Philippe, H., Hetrut, C., Hoffmann, J., and Bulet, P. (1996) J. Biol. Chem. 271, 21808-21813) and (ii) three new isoforms, mytilin C, D, and G1. The four molecules exhibited complementary antimicrobial properties. The cDNA sequence coding for the mytilin B precursor was obtained from a hemocyte cDNA library. This precursor contains a putative signal peptide of 22 residues, a processing peptide sequence of 34 amino acids, and a C-terminal extension of 48 residues rich in acidic residues. Distribution of mytilin B mRNA and of the corresponding peptide in various mussel tissues revealed that mytilins are synthesized and stored in a specific hemocyte subtype. Furthermore, in an experimental model of infection, we showed (i) a recruitment of hemocytes containing mytilins toward the injection site within hours following bacterial challenge, (ii) that mytilins probably play a prominent role in killing intracellular bacteria after phagocytosis, and (ii) later an increase of mytilin-like material occurred in the plasma suggesting a secondary systemic role.     

The caudal homeodomain protein activates Drosophila E2F gene expression

The Drosophila caudal homeobox gene is required for definition of the anteroposterior axis and for gut development, and CDX1 and CDX2, human homologs, play crucial roles in the regulation of cell proliferation and differentiation in the intestine. Most studies have indicated tumor suppressor functions of Cdx2, with inhibition of proliferation, while the effects of Cdx1 are more controversial. The influence of Drosophila Caudal on cell proliferation is unknown. In this study, we found three potential Caudal binding sequences in the 5′-flanking region of the Drosophila E2F (DE2F) gene and showed by transient transfection assays that they are involved in Caudal transactivation of the dE2F gene promoter. Analyses with transgenic flies carrying an E2F-lacZ fusion gene, with and without mutation in the Caudal binding site, indicated that the Caudal binding sites are required for expression of dE2F in living flies. Caudal-induced E2F expression was also confirmed with a GAL4-UAS system in living flies. In addition, ectopic expression of Caudal with heat-shock promotion induced melanotic tumors in larvae. These results suggest that Caudal is involved in regulation of proliferation through transactivation of the E2F gene in Drosophila.