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

<p>Abstract</p> <p>Background</p> <p>The planarian <it>Schmidtea mediterranea </it>and the cnidarian <it>Hydra vulgaris </it>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.</p> <p>Results</p> <p>This analysis revealed the induced expression of 27 genes in immune challenged <it>Schmidtea </it>and 35 genes in immune challenged <it>Hydra</it>. 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 <it>Schmidtea </it>and major vault protein in <it>Hydra</it>), stress responses (e.g. Hsp20 in <it>Schmidtea </it>and a PRP19/PSO4 DNA repair protein in <it>Hydra</it>), or to represent potential antimicrobial effectors (e.g. perforin-like protein in <it>Schmidtea </it>and PR-1-like protein and neutrophil cytosolic factor 1 in <it>Hydra</it>). As expected, septic wounding also induces expression of genes in <it>Schmidtea </it>and <it>Hydra </it>potentially involved in tissue remodeling associated with regeneration processes (e.g. matrix metalloproteinase in <it>Schmidtea </it>and a potential von Willebrand factor in <it>Hydra</it>).</p> <p>Conclusion</p> <p>We identified numerous immune-inducible genes in <it>Hydra </it>and <it>Schmidtea </it>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.</p

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

<p>Abstract</p> <p>Background</p> <p>The marine annelid <it>Platynereis dumerilii </it>(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.</p> <p>Results</p> <p>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 <it>P. dumerilii </it>are more related to counterparts from Deuterostomia than are those from Ecdysozoa, similarly as recently described for opsin and intron-rich genes.</p> <p>Conclusion</p> <p>Obtained results may allow for a better understanding of <it>Platynereis </it>immunity and support the view that <it>P. dumerilii </it>represents a suitable model for analyzing immune responses of Lophotrochozoa.</p

Identifizierung neuer Enzyme des Mevalonat-unabhängigen Methylerythritol-4-phosphat-Stoffwechselweges zur Isoprenoidbiosynthese

Der Mevalonat (MVA)-unabhängige Methylerythritol-4-phosphat (MEP)-Stoffwechselweg zur Biosynthese von Isoprenoiden wurde erst vor wenigen Jahren in Bakterien und in den pflanzlichen Plastiden identifiziert. Da dieser für viele Organismen essentielle Stoffwechselweg im Menschen nicht vorkommt, stellen die beteiligten Enzyme geeignete Zielstrukturen für die Entwicklung neuer antimikrobieller Wirkstoffe dar. Zu Beginn dieser Arbeit waren nur zwei Enzyme, die 1-Deoxy-D-xylulose-5-phosphat (DOXP)-Synthase (DXS) und DOXP-Reduktoisomerase (DXR) bekannt, die die ersten beiden Reaktionsschritte des MEP-Stoffwechselweges katalysieren. Mit Hilfe von bioinformatischen Ansätzen konnten verschiedene Gene identifiziert werden, die möglicherweise für noch unbekannte Enzyme des MEP-Stoffwechselweges kodieren. Um die Beteiligung dieser Gene an dem MEP-Stoffwechselweg zu beweisen, wurde ein genetischer Ansatz gewählt. Hierzu wurden zuerst genetisch veränderte Escherichia coli-Bakterien generiert, die in der Lage sind, Isopentenylpyrophosphat aus exogenem Mevalonat zu synthetisieren. Dazu wurde ein künstliches Operon konstruiert, das den E. coli-Bakterien die Expression der Hefe-Gene der MVA-Kinase (MVK), Phospho-MVA-Kinase (PMK) und der MVA-pyrophosphat-Decarboxylase (MPD) erlaubt. In diesen Bakterien konnten im MEP-Stoffwechselweg involvierte Gene deletiert werden. Dabei wurden die Gene gcpE und lytB als neue Gene des MEP-Stoffwechselweges in E. coli identifiziert. Zellen mit Deletionen dieser Gene konnten nur überleben, wenn das Medium mit Mevalonat supplementiert worden war oder die Zellen eine entsprechende episomale Kopie des jeweiligen Genes enthielten. Frühere Untersuchungen haben gezeigt, daß ein bisher unbekanntes Intermediat des MEP-Stoffwechselweges zur Aktivierung von polyklonalen Vg9Vd2 T-Zellen des menschlichen Immunsystems verantwortlich ist. Deshalb wurden die generierten Deletionsmutanten in gd T-Zellaktivierungs-Experimente eingesetzt. Hierbei stellte sich heraus, daß gcpE-Gendeletionsmutanten ihr gd T-Zellaktivierungspotential fast vollständig verloren hatten, während lytB-Gendeletionsmutanten ein über 100-fach höheres Potential als der Wildtyp besaßen. Als gd T-Zellaktivator konnte eine Substanz isoliert werden, deren Struktur als (E)-4-Hydroxy-3-methyl-but-2-enyl-pyrophosphat (HMBPP) aufgeklärt wurde. Diese Ergebnisse legen nahe, daß HMBPP ein neues Intermediat des MEP-Stoffwechselweges ist und das Produkt von GcpE und das Substrat von LytB darstellt

Stronger Food and Nutrition Security Impacts from More Intense Project Participation: Evidence from a Multi-Country Intervention Program

Rigorous experiments show that nutrition-sensitive intervention programs can contribute to improved food and nutrition security (FNS) of rural households in low-and middle-income countries. Targeted individuals may, however, choose to engage with the intervention package at different intensities. It is yet unclear to what extent individual participation in more interventions influences FNS outcomes. Positive links would justify efforts by development stakeholders to diversify intervention packages and enable, encourage, or incentivize beneficiaries to participate in many different interventions. Using cross-sectional data from 2733 households across seven countries, we first estimate effects of a multi-sectoral intervention program using probit regressions, propensity score matching, and inverse probability weighted regression adjustment. Over the course of the three-year program, beneficiaries joined 8.3 interventions, on average. We find that targeted households were 6–9 percent more likely to be food secure, and targeted women and children were 15–17 percent more likely to consume a nutrient-adequate diet. Our estimates show that, across three indicators of FNS, each additional intervention increased the probability of achieving positive outcomes by about 1 percent. We conclude that investments in diversified intervention programs can be justified by stronger FNS benefits. Development stakeholders could enable strong individual participation by reducing transaction and opportunity costs of participation.Peer Reviewe

MMPs Regulate both Development and Immunity in the Tribolium Model Insect

BACKGROUND: Matrix metalloproteinases (MMPs) are evolutionarily conserved and multifunctional effector molecules in development and homeostasis. In spite of previous, intensive investigation in vitro and in cell culture, their pleiotrophic functions in vivo are still not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We show that the genetically amenable beetle Tribolium castaneum represents a feasible model organism to explore MMP functions in vivo. We silenced expression of three insect-type Tribolium MMP paralogs and their physiological inhibitors, TIMP and RECK, by dsRNA-mediated genetic interference (RNAi). Knock-down of MMP-1 arrested development during pupal morphogenesis giving phenotypes with altered antennae, compound eyes, wings, legs, and head. Parental RNAi-mediated knock-down of MMP-1 or MMP-2 resulted in larvae with non-lethal tracheal defects and with abnormal intestines, respectively, implicating additional roles of MMPs during beetle embryogenesis. This is different to findings from the fruit fly Drosophila melanogaster, in which MMPs have a negligible role in embryogenesis. Confirming pleiotrophic roles of MMPs our results also revealed that MMPs are required for proper insect innate immunity because systemic knock-down of Tribolium MMP-1 resulted in significantly higher susceptibility to the entomopathogenic fungus Beauveria bassiana. Moreover, mRNA levels of MMP-1, TIMP, and RECK, and also MMP enzymatic activity were significantly elevated in immune-competent hemocytes upon stimulation. To confirm collagenolytic activity of Tribolium MMP-1 we produced and purified recombinant enzyme and determined a similar collagen IV degrading activity as observed for the most related human MMP, MMP-19. CONCLUSIONS/SIGNIFICANCE: This is the first study, to our knowledge, investigating the in vivo role of virtually all insect MMP paralogs along with their inhibitors TIMP and RECK in both insect development and immunity. Our results from the Tribolium model insect indicate that MMPs regulate tracheal and gut development during beetle embryogenesis, pupal morphogenesis, and innate immune defense reactions thereby revealing the evolutionarily conserved roles of MMPs

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

<p>Abstract</p> <p>Background</p> <p>The larvae of the greater wax moth <it>Galleria mellonella </it>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 <it>Galleria</it>, we subjected the transcriptome of different developmental stages and immune-challenged larvae to next generation sequencing.</p> <p>Results</p> <p>We performed a <it>Galleria </it>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 (<it>E </it>≤ 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.</p> <p>Conclusion</p> <p>Here, we have developed extensive transcriptomic resources for <it>Galleria</it>. The data obtained is rich in gene transcripts related to immunity, expanding remarkably our knowledge about immune and stress-inducible genes in <it>Galleria </it>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.</p

Immunity and other defenses in pea aphids, Acyrthosiphon pisum

The genome of the pea aphid Acyrthosiphon pisum lacks genes thought to be crucial in other insects for recognition, signaling and killing of microbes

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 (IC50 value ∼1 μM) 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 Ti 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 μM. 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

Analysis of the immune-inducible transcriptome from microbial stress resistant, rat-tailed maggots of the drone fly <it>Eristalis tenax</it>

Abstract 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.</p