Crystal Structure of Diedel, a Marker of the Immune Response of Drosophila melanogaster

Background: The Drosophila melanogaster gene CG11501 is up regulated after a septic injury and was proposed to act as a negative regulator of the JAK/STAT signaling pathway. Diedel, the CG11501 gene product, is a small protein of 115 residues with 10 cysteines. Methodology/Principal Findings: We have produced Diedel in Drosophila S2 cells as an extra cellular protein thanks to its own signal peptide and solved its crystal structure at 1.15 A ˚ resolution by SIRAS using an iodo derivative. Diedel is composed of two sub domains SD1 and SD2. SD1 is made of an antiparallel b-sheet covered by an a-helix and displays a ferredoxin-like fold. SD2 reveals a new protein fold made of loops connected by four disulfide bridges. Further structural analysis identified conserved hydrophobic residues on the surface of Diedel that may constitute a potential binding site. The existence of two conformations, cis and trans, for the proline 52 may be of interest as prolyl peptidyl isomerisation has been shown to play a role in several physiological mechanisms. The genome of D. melanogaster contains two other genes coding for proteins homologous to Diedel, namely CG43228 and CG34329. Strikingly, apart from Drosophila and the pea aphid Acyrthosiphon pisum, Diedel-related sequences were exclusively identified in a few insect DNA viruses of the Baculoviridae and Ascoviridae families. Conclusion/Significance: Diedel, a marker of the Drosophila antimicrobial/antiviral response, is a member of a small famil

Die induzierbare antivirale Immunantwort von Drosophila melanogaster

In der vorliegenden Arbeit wurde Drosophila melanogaster als Modell ge- nutzt, um die angeborene Immunantwort gegen virale Infektionen zu studie- ren. Wir untersuchten mit Hilfe von genomweiten microarrays das Transkriptom von Fliegen, welche entweder mit dem Drosophila C Virus (DCV), dem Flock- house Virus (FHV) oder dem Sindbis Virus (SINV) infiziert waren. Infektion mit diesen drei positiv orientierten Einzelstrang RNS Viren führte zu einer starken transkriptionellen Antwort, welche deutlich virusspezifische Indukti- onsmuster zeigte. Um die biologische Signifikanz dieser beobachteten Muster zu studieren, testeten wir Mutanten verschiedener Signalwege, nämlich den Imd, den Toll und den Jak-STAT Signalweg, für Suszebtibiltät nach viraler Infektion. Wir führten eine Vergleichsstudie durch, in welcher wir ein Set von 5 verschiedenen RNS Viren benutzten. Das Set beinhaltete die positiv ori- entierten RNS Viren DCV, Cricket Paralysis Virus (CrPV), FHV und SINV und den negativ orientierten RNS Virus Vesicular Stomatis Virus (VSV). Die Dicistroviren DCV und CrPV formten eine spezielle Gruppe unter den getesteten Viren, da Mutanten für den Jak-STAT und den Toll Signalweg anfällig für DCV und CrPV Infektion waren. Mutanten starben früher als Wildtyp Fliegen und zeigten erhöhte virale Titer. Im Gegensatz dazu, sa- hen wir keinen Phänotyp, weder im Überleben noch in den viralen Titern nach Infektion mit FHV, SINV oder VSV. Der Imd Signalweg wurde nicht als eine bedeutende Komponente der antiviralen Immunantwort identifiziert. Trotzdem zeigten Mutanten dieses Signalweges eine Tendenz zu reduzierten viralen Titern nach Dicistrovirus Infektion. Wir zeigten weiterhin, dass re- lativ gesehen, der Jak-STAT Signalweg genauso bedeutend für die Abwehr gegen Dicistriviren ist wie RNS Interferenz. Diese Daten unterstreichen die Wichtigkeit der induzierbaren Antwort in Drosophila. Wir charakterisierten Chifoumi (cfm ) alias CG11501 als ein Beispiel für ein virusinduziertes Gen. Wir zeigten, dass cfm ein zirkulierendes Peptid ko- diert, welches nach viraler Infektion im Fettkörper exprimiert und in den He- molymph sekretiert wird. Mutanten für dieses Gen, zeigten reduzierte virale Titer und verlängertes Überleben nach Infektion mit DCV. Diese Ergebnisse legen Nahe, dass CFM möglicherweise ein wirtskodierter Faktor ist, welcher die virale Replikation in Drosophila fördert.The work presented in this thesis used the fruit fly Drosophila melanogaster as a model system to study the inducible innate immune response against virus infection. We studied the transcriptome of flies infected with either Drosophila C virus (DCV), Flockhouse virus (FHV) or Sindbis virus (SINV) using genome-wide microarray analysis. Infection with these postive-sense single-stranded RNA viruses triggered a strong inducible response that showed evidence of virus- specific induction patterns. In order to study the biological significance of these gene inductions, we investigated susceptibility to viral infection of mu- tant flies for signaling pathways, such as the Imd, the Toll and the Jak-STAT pathway. We conducted a comparative analysis using a set of five distinct RNA viruses, that included the positive-sense RNA viruses DCV, Cricket paralysis virus (CrPV), FHV and SINV, and the negative-sense RNA virus Vesicular stomatis virus (VSV). We found that the Dicistroviruses DCV and CrPV formed a unique group among the viruses tested, as mutants of both, the Jak-STAT and the Toll pathway were susceptible to infection with DCV and CrPV. The observed susceptibility correlated with an increase in viral titers. No phenotype, neither in viral load nor in survival susceptibility was observed upon infection with FHV, SINV or VSV. We found no ma jor role for the Imd pathway in the antiviral defense, however, mutant flies of this pathway showed a trend towards a reduction in Dicistrovirus load. Further- more, we showed that the Jak-STAT pathway is as important for the defense against Dicistrovirus infection as RNA interference, highlighting the relative importance of the inducible response against this virus family. As an example for a virus-induced gene, we characterized Chifoumi (cfm ) alias CG11501 . We showed that cfm codes for a circulating peptide that is induced in the fat body upon viral infection and secreted in the hemolymph. cfm mutant flies showed decreased viral titers and prolonged survival upon infection with DCV, suggesting that CFM may function as a host-encoded factor that propagates viral replication in drosophila

Antiviral immunity in drosophila

Genetic analysis of the drosophila antiviral response indicates that RNA interference plays a major role. This contrasts with the situation in mammals, where interferon-induced responses mediate innate antiviral host-defense. An inducible response also contributes to antiviral immunity in drosophila, and similarities in the sensing and signaling of viral infection are becoming apparent between drosophila and mammals. In particular, DExD/H box helicases appear to play a crucial role in the cytosolic detection of viral RNAs in flies and mammals

A plant-like mitochondrial carrier family protein facilitates mitochondrial transport of di- and tricarboxylates in Trypanosoma brucei

The procyclic form of the human parasite Trypanosoma brucei harbors one single, large mitochondrion containing all tricarboxylic acid (TCA) cycle enzymes and respiratory chain complexes present also in higher eukaryotes. Metabolite exchange among subcellular compartments such as the cytoplasm, the mitochondrion, and the peroxisomes is crucial for redox homeostasis and for metabolic pathways whose enzymes are dispersed among different organelles. In higher eukaryotes, mitochondrial carrier family (MCF) proteins transport TCA-cycle intermediates across the inner mitochondrial membrane. Previously, we identified several MCF members that are essential for T. brucei survival. Among these, only one MCF protein, TbMCP12, potentially could transport dicarboxylates and tricarboxylates. Here, we conducted phylogenetic and sequence analyses and functionally characterised TbMCP12 in vivo. Our results suggested that similarly to its homologues in plants, TbMCP12 transports both dicarboxylates and tricarboxylates across the mitochondrial inner membrane. Deleting this carrier in T. brucei was not lethal, while its overexpression was deleterious. Our results suggest that the intracellular abundance of TbMCP12 is an important regulatory element for the NADPH balance and mitochondrial ATP-production

A label-free aptamer-based nanogap capacitive biosensor with greatly diminished electrode polarization effects

A significant impediment to the use of impedance spectroscopy in bio-sensing is the electrode polarization effect that arises from the movement of free ions to the electrode-solution interface, forming an electrical double layer (EDL). The EDL screens the dielectric response of the bulk and its large capacitance dominates the signal response at low frequency, masking information particularly relevant for biological samples, such as molecular conformation changes and DNA hybridization. The fabrication of nanogap capacitors with electrode separation less than the EDL thickness can significantly reduce electrode polarization effects and provide enormous improvement in sensitivity due to better matching of the sensing volume with the size of the target entities. We report on the fabrication of a horizontal thin-film nanogap capacitive sensor with electrode separation of 40 nm that shows almost no electrode polarization effects when measured with water and ionic buffer solutions, thereby allowing direct quantification of their relative permittivity at low frequencies. Surface modification of the electrodes with thiol-functionalized single strand DNA aptamers transforms the device into a label-free biosensor with high sensitivity and selectivity towards the detection of a specific protein. Using this approach, we have developed a biosensor for the detection of human alpha thrombin. In addition, we also examine frequency dependent permittivity measurements on high ionic strength solutions contained within the nanogap and discuss how these support recent experimental observations of large Debye lengths. A large shift in the Debye relaxation frequency to lower frequency is also found, which is consistent with water molecules being in a rigid-like state, possibly indicating the formation of an ordered "ice-like" phase. Altogether, this work highlights the need for better understanding of fluids in confined, nanoscale geometries, from which important new applications in sensing may arise

Improved DNA extraction efficiency from low level cell numbers using a silica monolith based micro fluidic device

The evaluation of a micro fluidic system with an integrated silica monolith for performing DNA extraction from limited biological samples has been carried out. Low DNA target concentrations usually require the addition of carrier RNA to ensure desired extraction efficiencies. Here, we demonstrate a micro fluidic extraction system with increasingly efficient extraction performances for biological samples containing <15ng of total DNA without the need of adding carrier nucleic acids. All extracted DNA showed successful amplification via the polymerase chain reaction demonstrating both the effectiveness of the proposed system at removing potential inhibitors and yielding good quality DNA. The work presented here beneficially identifies reduced sample volumes/concentrations as suitable for processing with respect to downstream analysis by enabling pre-concentration of the biological sample, particularly important when dealing with clinical or forensic specimens

Integrated DNA extraction and amplification using electrokinetic pumping in a microfluidic device

An integrated system employing anion exchange for the extraction of DNA from biological samples prior to polymerase chain reaction DNA amplification has been developed, based on microfluidic methodology utilising electrokinetic pumping. In this system, the biological samples were added directly to chitosan-coated silica beads to facilitate DNA immobilisation. The purified, pre-concentrated DNA was then eluted using a combination of electro-osmotic flow enhanced with electrophoretic mobility, which enable DNA to be transported by both mechanisms into the DNA amplification chamber. Through optimisation of the DNA elution conditions, average DNA extraction efficiencies of 69.1% were achievable. Subsequent DNA amplification performed on the microfluidic system demonstrated not only the ability to use electrokinetic movement to integrate the two processes on a single device, but also that the quality and quantity of DNA eluted was suitable for downstream analysis. This work offers an attractive real-world to chip interface and a route to simpler Lab-on-a-Chip technology which eliminates the need for moving parts

Phylogenetic analysis of <i>Diedel</i>.

<p>(<b>A</b>) Genome localization of <i>Diedel</i> and related genes in drosophilids. Genes are named according to Flybase. The asterisk indicates that sequence has been been re-analysed and differs from the annotated one. For each <i>Drosophila</i> species are indicated the orientation and the position of the gene or on the chromosome (3R, X, 2) or on a scaffold. The precise position is indicated by the number at the beginning and the end of each base on the sequence given on fly base. When the distances are not too long, the position respect a scale (for pseudoobscura, persimilis and virilis). In the case of virilis, the gene GJ11856 is duplicated. No new ID has been proposed. (<b>B</b>) Phylogeny of Diedel-related molecules. The proteins are named according to Flybase for Drosophila species. IDs of the viral molecules can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416.s001" target="_blank">Text S1</a>. In red the melanogaster subgroup, in green the obscura group, in blue the repleta and the virilis groups, in purple the sequences from viruses. The sequences are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416.s001" target="_blank">Text S1</a>.</p

Amino acid sequence alignment of Driedel and other homolous proteins.

<p>The Diedel protein of <i>Drosophila melanogaster</i> was aligned to homologous gene products from <i>Drosophila melanogaster</i> (CG34329) and <i>Pseudalatia unipuncta</i> granulovirus (PuGV). The numbering is that of Diedel in this study. Secondary structure elements, <i>i.e.</i> strands and helices, are indicated below the sequences as arrows and rectangles, respectively. Conserved residues are boxed, and strictly conserved residues are shown in white with a red background. Note that the level of sequence identity is much more higher in the SD2 sub-domain than in the SD1 sub-domain. The figure was generated with ESPript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033416#pone.0033416-Gouet1" target="_blank">[36]</a>.</p