Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.

Symbiont-mediated protection against fungal pathogens in pea aphids: a role for pathogen specificity?

Here we show that a bacterial endosymbiont, Regiella insecticola, protects pea aphids (Acyrthosiphon pisum) from the aphid-specific fungal entomopathogen Zoophthora occidentalis but not from the generalist insect fungal pathogen Beauveria bassiana. This finding highlights the complex influence of fungi on the dynamics of this economically important agricultural pest.

Horizontally transferred fungal carotenoid genes in the two-spotted spider mite Tetranychus urticae.

Carotenoids are organic pigments commonly synthesized by plants, algae and some micro-organisms. Through absorption of light energy, carotenoids facilitate photosynthesis and provide protection against photo-oxidation. While it was presumed that all carotenoids in animals were sequestered from their diets, aphids were recently shown to harbour genomic copies of functional carotenoid biosynthesis genes that were acquired via horizontal gene transfer from fungi. Our search of available animal transcripts revealed the presence of two related genes in the two-spotted spider mite Tetranychus urticae. Phylogenetic analyses suggest that the T. urticae genes were transferred from fungi into the spider mite genome, probably in a similar manner as recently suggested for aphids. The genes are expressed in both green and red morphs, with red morphs exhibiting higher levels of gene expression. Additionally, there appear to be changes in the expression of these genes during diapause. As carotenoids are associated with diapause induction in these animals, our results add to recent findings highlighting the importance of eukaryotic horizontal gene transfer in the ecology and evolution of higher animals.

A comprehensive transcriptome and immune-gene repertoire of the lepidopteran model host Galleria mellonella.

BACKGROUND: The larvae of the greater wax moth Galleria mellonella are increasingly used (i) as mini-hosts to study pathogenesis and virulence factors of prominent bacterial and fungal human pathogens, (ii) as a whole-animal high throughput infection system for testing pathogen mutant libraries, and (iii) as a reliable host model to evaluate the efficacy of antibiotics against human pathogens. In order to compensate for the lack of genomic information in Galleria, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing. RESULTS: We performed a Galleria transcriptome characterization on the Roche 454-FLX platform combined with traditional Sanger sequencing to obtain a comprehensive transcriptome. To maximize sequence diversity, we pooled RNA extracted from different developmental stages, larval tissues including hemocytes, and from immune-challenged larvae and normalized the cDNA pool. We generated a total of 789,105 pyrosequencing and 12,032 high-quality Sanger EST sequences which clustered into 18,690 contigs with an average length of 1,132 bases. Approximately 40% of the ESTs were significantly similar (E ≤ e-03) to proteins of other insects, of which 45% have a reported function. We identified a large number of genes encoding proteins with established functions in immunity related sensing of microbial signatures and signaling, as well as effector molecules such as antimicrobial peptides and inhibitors of microbial proteinases. In addition, we found genes known as mediators of melanization or contributing to stress responses. Using the transcriptomic data, we identified hemolymph peptides and proteins induced upon immune challenge by 2D-gelelectrophoresis combined with mass spectrometric analysis. CONCLUSION: Here, we have developed extensive transcriptomic resources for Galleria. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in Galleria and providing the complete sequences of genes whose primary structure have only partially been characterized using proteomic methods. The generated data provide for the first time access to the genetic architecture of immunity in this model host, allowing us to elucidate the molecular mechanisms underlying pathogen and parasite response and detailed analyses of both its immune responses against human pathogens, and its coevolution with entomopathogens.

Molecular characterization and expression patterns of heat shock proteins in Spodoptera littoralis, heat shock or immune response?

The Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.), is a major agricultural lepidopterous pest causing extensive damage in a variety of crops including vegetable, cotton, fodder, and fiber crops. Heat shock protein (HSP) family members play important roles in protecting insects against environmental stressors. In this study, we characterized three putative heat shock proteins (SpliHsp70, SpliHsp90, and SpliHSF) from S. littoralis and analyzed their expression levels in response to heat, cold, ultraviolet irradiation, Bacillus thuringiensis, and Spodoptera littoralis nucleopolyhedrovirus treatments. Significant upregulation of SpliHsp70 was observed in female pupae, while the highest expression levels of SpliHsp90 and SpliHSF were found in female adults. Heat shock triggered increases in SpliHsp levels compared to cold treatment. SpliHsp90 exhibited the highest expression levels during the first 30 min of UV treatment. Both bacterial and viral pathogenic agents effected the regulation of Hsps in S. littoralis. These findings suggest that SpliHsp genes might play significant roles in the response to biotic and abiotic stress, as well as in the regulation of developmental stages.

Galleria mellonella as a model system for studying Listeria pathogenesis.

Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37 degrees C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L. monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.

Host-derived extracellular nucleic acids enhance innate immune responses, induce coagulation, and prolong survival upon infection in insects.

Extracellular nucleic acids play important roles in human immunity and hemostasis by inducing IFN production, entrapping pathogens in neutrophil extracellular traps, and providing procoagulant cofactor templates for induced contact activation during mammalian blood clotting. In this study, we investigated the functions of extracellular RNA and DNA in innate immunity and hemolymph coagulation in insects using the greater wax moth Galleria mellonella a reliable model host for many insect and human pathogens. We determined that coinjection of purified Galleria-derived nucleic acids with heat-killed bacteria synergistically increases systemic expression of antimicrobial peptides and leads to the depletion of immune-competent hemocytes indicating cellular immune stimulation. These activities were abolished when nucleic acids had been degraded by nucleic acid hydrolyzing enzymes prior to injection. Furthermore, we found that nucleic acids induce insect hemolymph coagulation in a similar way as LPS. Proteomic analyses revealed specific RNA-binding proteins in the hemolymph, including apolipoproteins, as potential mediators of the immune response and hemolymph clotting. Microscopic ex vivo analyses of Galleria hemolymph clotting reactions revealed that oenocytoids (5-10% of total hemocytes) represent a source of endogenously derived extracellular nucleic acids. Finally, using the entomopathogenic bacterium Photorhabdus luminescens as an infective agent and Galleria caterpillars as hosts, we demonstrated that injection of purified nucleic acids along with P. luminescens significantly prolongs survival of infected larvae. Our results lend some credit to our hypothesis that host-derived nucleic acids have independently been co-opted in innate immunity of both mammals and insects, but exert comparable roles in entrapping pathogens and enhancing innate immune responses.

Identification of collagen IV derived danger/alarm signals in insect immunity by nanoLC-FTICR MS.

The immune system can be stimulated by microbial molecules as well as by endogenously derived danger/alarm signals of host origin. Using the lepidopteran model insect Galleria mellonella, we recently discovered that fragments of collagen IV, resulting from hydrolysis by microbial metalloproteinases, represent danger/alarm signals in insects. Here, we characterized immune-stimulatory peptides generated by thermolysin-mediated degradation of collagen IV using nanospray ionization Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) after separation by nanoscale liquid chromatography (nanoLC). The combination of FTICR MS analysis and de novo peptide sequencing resulted in the identification of 38 specific collagen IV fragments of which several peptides included the integrin-binding motif RGD/E known from numerous mammalian immune-related proteins. Custom-synthesized peptides corresponding either to the presently identified collagen peptide GIRGEHyp or to a well-known integrin-binding RGD peptide (GRGDS) were injected into G. mellonella to determine their immune-stimulatory activities in vivo. Both peptides stimulated immune cells and systemically the expression of lysozyme and a specific inhibitor of microbial metalloproteinases. Further examination using specific MAP kinase inhibitors indicated that MEK/ERK and p38 are involved in RGD/E-mediated immune-signaling pathways, whereas JNK seems to play only a minor role.

Insect peptide metchnikowin confers on barley a selective capacity for resistance to fungal ascomycetes pathogens.

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC(50) value approximately 1 muM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the T(i) plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 muM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

Identification of a lepidopteran matrix metalloproteinase with dual roles in metamorphosis and innate immunity.

Matrix metalloproteinases (MMPs) are key enzymes in mammalian tissue remodeling and inflammation. Recently, we postulated that an endogenous MMP expressed in the lepidopteran model Galleria mellonella during metamorphosis causes degradation of collagen-IV, which in turn results in activation of innate immunity. Here, we report that degradation of collagen-IV by hemocytes is enhanced upon injection of bacterial lipopolysaccharide (LPS), and that this activity is sensitive to the MMP-inhibitor GM6001. Therefore, we screened for enzymes behind this activity and identified the first MMP from Lepidoptera (Gm1-MMP), and the third from insects. Gm1-MMP shares homology with the first MMP from Drosophila (Dm1-MMP) known to be essential for tissue remodeling during metamorphosis. Using quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis, we confirmed up-regulation of Gm1-MMP expression after pupation, when extracellular matrix breakdown of larval tissues occurs. In addition, we determined that LPS challenge induces Gm1-MMP expression in hemocytes, implicating its participation in collagen-IV degradation upon septic injury. These results suggest dual roles of Gm1-MMP in innate immunity and metamorphosis. Interestingly, our phylogenetic analysis elucidates that Gm1-MMP share highest similarity with human MMP-19 and MMP-28, whose functions in mammalian wounding and inflammatory response have recently been demonstrated; hence, the present findings may provide insights into the evolutionarily conserved features of MMPs.

Identification of immune inducible genes from the velvet worm Epiperipatus biolleyi (Onychophora).

Onychophora are the next relatives of Arthropoda and, hence, represent an important taxon to unravel relationships among Insecta, Crustacea, Arachnida, and Myriapoda. Here, we screened for immune inducible genes from the onychophoran Epiperipatus biolleyi (Peripatidae) by injecting crude bacterial LPS and applying the suppression subtractive hybridization technique. Our analysis of 288 cDNAs resulted in identification of 36 novel genes in E. biolleyi whose potential homologues from other animals are known to mediate immune-related signaling (e.g. mitogen-activated protein kinase kinase 1 and immunoglobulin enhancer binding protein), to be involved in cellular processes (e.g. perilipin and myosin light chain), or to act as immune effector molecules (e.g. lysosomal beta-galactosidase, a putative antimicrobial peptide and a potential thiolester containing protein). Comparisons with homologous genes from other animals including the two most favored ecdysozoan model organisms of innate immunity research, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, provide further insights into the origin and evolution of Arthropoda immunity.

Beetle immunity: Identification of immune-inducible genes from the model insect Tribolium castaneum.

The red flour beetle, Tribolium castaneum, is an established genetically tractable model insect for evolutionary and developmental studies. Therefore, it may also represent a valuable model for comparative analysis of insect immunity. Here, we used the suppression subtractive hybridization method to identify Tribolium genes that are transcriptionally induced in response to injection of crude lipopolysaccharide (LPS). Determined genes encode proteins that share sequence similarities with counterparts from other insects known to mediate sensing of infection (e.g. Toll and PGRP) or to represent potential antimicrobial effectors (e.g. ferritin, c-type lysozyme, serine proteinase inhibitors, and defensins). Especially significant is the identification of thaumatin-like peptides, representing ancient antifungal peptides originally reported from plants, that are absent from the genomes of many other insects such as Drosophila, Anopheles, and Apis. We produced recombinant thaumatin-1 in bacteria and we found that it represents an antimicrobial peptide against filamentous fungi in Tribolium. Additionally, septic injury induces expression of genes involved in stress adaptation (e.g. heat-shock proteins) or insecticide resistance (e.g. cytochrome P450s) in Tribolium, suggesting that there may be crosstalk between the immune and stress responses.

Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris.

BACKGROUND: The planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris have emerged as valuable model organisms in regeneration and stem cell research because of their prominent ability to regenerate a complete organism from any small body fragment. Under natural conditions wounding may result from predator attacks. These injuries open their innermost to a wide array of microbes present in the environment. Therefore, we established the hypothesis that regeneration processes may be linked to or at least accompanied by innate immune responses. In order to screen for septic wounding inducible genes we dissected individuals using a scalpel in the presence of a crude bacterial lipopolysaccharide preparation that is commonly used to elicit innate immune responses in animals and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes. RESULTS: This analysis revealed the induced expression of 27 genes in immune challenged Schmidtea and 35 genes in immune challenged Hydra. Identified genes from both animals encode proteins that share sequence similarities with potential homologues from other organisms known to be involved in signaling (e.g. calreticulin in Schmidtea and major vault protein in Hydra), stress responses (e.g. Hsp20 in Schmidtea and a PRP19/PSO4 DNA repair protein in Hydra), or to represent potential antimicrobial effectors (e.g. perforin-like protein in Schmidtea and PR-1-like protein and neutrophil cytosolic factor 1 in Hydra). As expected, septic wounding also induces expression of genes in Schmidtea and Hydra potentially involved in tissue remodeling associated with regeneration processes (e.g. matrix metalloproteinase in Schmidtea and a potential von Willebrand factor in Hydra). CONCLUSION: We identified numerous immune-inducible genes in Hydra and Schmidtea that show a similar distribution corresponding to their physiological roles, although lineages of both animals split from their common ancestor for more than five hundred millions of years. The present study is the first analysis of immune-inducible genes of these two phylogenetically distant model organisms of regeneration and provide numerous candidate genes that we can use as a starting point for comparative examination of interrelationships between immunity and homeostasis.

Dissemination of Fusarium proliferatum by mealworm beetle Tenebrio molitor.

BACKGROUND: Plant pathogenic fungi of the genus Fusarium infect a wide array of crops and produce numerous health-threatening mycotoxins. Recently, we found that larvae of the common pest of stored products Tenebrio molitor preferably fed on grains colonized with Fusarium proliferatum. We draw the hypothesis that the increased attractiveness of infected grains for mealworms facilitates dispersal of the fungus. In this work we examined the dissemination of F. proliferatum and further Fusarium spp. by adults of T. molitor. RESULTS: Mealworm beetle Tenebrio molitor transmitted Fusarium species F. avenaceum, F. culmorum, F. poae, and F. proliferatum to wheat grains with varying efficiency. F. proliferatum was disseminated most efficiently: 20 days after feeding on Fusarium cultures, the beetles still transmitted F. proliferatum to most grains exposed to feeding. The transmission of F. culmorum gradually declined over time and the transmission of the other Fusarium spp. ceased completely 20 d after beetles feeding of fungal cultures. Propagules of F. proliferatum and F. culmorum were traceable in beetles' feces for 20 days while no colonies of F. poae and F. avenaceum were detectable after 5 days. Because F. proliferatum was transmitted by mealworms most efficiently, this species was further investigated. Mealworm beetles T. molitor preferred feeding on grains colonized with F. proliferatum as compared to uninfected grains. Male beetles infected with F. proliferatum transmitted the fungus by copulation. CONCLUSIONS: Efficient dissemination of F. proliferatum by mealworm beetle together with the feeding preference of the beetle for grains colonized with F. proliferatum show that the chemical phenotype of the fungus responsible for the enhanced attractiveness of infected grains is subjected to positive selection. This indicates that adaptation of F. proliferatum to transmission by insects involved an alteration of insects' feeding preferences.

Analysis of the immune-inducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly Eristalis tenax.

BACKGROUND: The saprophagous and coprophagous maggots of the drone fly Eristalis tenax (Insecta, Diptera) have evolved the unique ability to survive in aquatic habitats with extreme microbial stress such as drains, sewage pools, and farmyard liquid manure storage pits. Therefore, they represent suitable models for the investigation of trade-offs between the benefits resulting from colonization of habitats lacking predators, parasitoids, or competitors and the investment in immunity against microbial stress. In this study, we screened for genes in E. tenax that are induced upon septic injury. Suppression subtractive hybridization was performed to selectively amplify and identify cDNAs that are differentially expressed in response to injected crude bacterial endotoxin (LPS). RESULTS: Untreated E. tenax maggots exhibit significant antibacterial activity in the hemolymph which strongly increases upon challenge with LPS. In order to identify effector molecules contributing to this microbial defense we constructed a subtractive cDNA library using RNA samples from untreated and LPS injected maggots. Analysis of 288 cDNAs revealed induced expression of 117 cDNAs corresponding to 30 novel gene clusters in E. tenax. Among these immune-inducible transcripts we found homologues of known genes from other Diptera such as Drosophila and Anopheles that mediate pathogen recognition (e.g. peptidoglycan recognition protein) or immune-related signaling (e.g. relish). As predicted, we determined a high diversity of novel putative antimicrobial peptides including one E. tenax defensin. CONCLUSION: We identified 30 novel genes of E. tenax that were induced in response to septic injury including novel putative antimicrobial peptides. Further analysis of these immune-related effector molecules from Eristalis may help to elucidate the interdependency of ecological adaptation and molecular evolution of the innate immunity in Diptera.

Identification of immune-related genes from an apterygote insect, the firebrat Thermobia domestica.

In this study, we report the analysis of the immune-related transcriptome from an apterygote insect, the firebrat Thermobia domestica (Zygentoma, Lepismatidae), which currently emerges as a suitable model insect for evolutionary and developmental studies. The suppression subtractive hybridization method was used for targeted screening of genes that are up-regulated in response to injected bacterial lipopolysaccharide (LPS). A subtracted cDNA library enriched in immune-inducible genes was constructed and analysis of 288 cDNAs resulted in identification of 26 novel genes in T. domestica. Among these immune-related transcripts we found homologues of genes from other insects which are involved in the regulation of signaling cascades and six novel putative antimicrobial peptides. The identified genes implicate the presence of sophisticated regulatory mechanisms in insect immune signaling and give insight into evolutionarily conserved features of insect innate immunity.

Analysis of the immune-related transcriptome of a lophotrochozoan model, the marine annelid Platynereis dumerilii.

BACKGROUND: The marine annelid Platynereis dumerilii (Polychaeta, Nereididae) has been recognized as a slow-evolving lophotrochozoan that attracts increasing attention as a valuable model for evolutionary and developmental research. Here, we analyzed its immune-related transcriptome. For targeted identification of immune-induced genes we injected bacterial lipopolysaccharide, a commonly used elicitor of innate immune responses, and applied the suppression subtractive hybridization technique that selectively amplifies cDNAs of differentially expressed genes. RESULTS: Sequence analysis of 288 cDNAs revealed induced expression of numerous genes whose potential homologues from other animals mediate recognition of infection (e.g. complement receptor CD35), signaling (e.g. myc and SOCS), or act as effector molecules like ferritins and the bactericidal permeability-increasing protein. Interestingly, phylogenetic analyses implicate that immune-related genes identified in P. dumerilii are more related to counterparts from Deuterostomia than are those from Ecdysozoa, similarly as recently described for opsin and intron-rich genes. CONCLUSION: Obtained results may allow for a better understanding of Platynereis immunity and support the view that P. dumerilii represents a suitable model for analyzing immune responses of Lophotrochozoa.

A positively charged cluster in the epidermal growth factor-like domain of Factor VII-activating protease (FSAP) is essential for polyanion binding.

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Galleria mellonella.

A novel link between development and immunity in insects is introduced. Transiently enhanced expression of lysozyme, gallerimycin and the insect metalloproteinase inhibitor was discovered at the onset of metamorphosis of the greater wax moth, Galleria mellonella. Relative quantification of mRNAs encoding for these antimicrobial peptides using real-time PCR documents their induced expression during transformation of last instar larvae into prepupae and upon injection of either recombinant interstitial collagenase (MMP-1) or small-sized fragments of collagen type IV. The latter were also found to stimulate both nuclear import of c-Rel-proteins in the fat body, implicating activation of Toll or Imd-related signaling pathways, and subsequent synthesis of antimicrobial peptides. Obtained results implicate that degradation of collagen-IV by either microbial metalloproteinases associated with invading pathogens or endogenous matrix metalloproteinases contributing to degradation of extracellular matrix during metamorphosis stimulate innate immune responses.

LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis.

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C. The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM.