The fruit fly Drosophila melanogaster is recognised as the most widely established genetic model of immunity of the contemporary scientific era, exhibiting a high degree of conservation between Drosophila and mammalian innate immunity genes. However, whilst the majority of Drosophila immunity studies have previously been performed in adults and larvae, the embryo has recently emerged as a potentially viable model system; aiding in vivo studies and providing a more amendable system to undertake live imaging, hence evading many of the caveats associated with current immunity models. This project aimed to further develop the Drosophila embryo as a more potent and insightful immunity model, focusing on the immune response to bacterial infection. Initial results demonstrated that the Stage 15 Drosophila embryo was able to mount a relatively robust immune response to bacterial infection. This included induction of antimicrobial peptide (AMP) genes upon a range of bacterial stimuli; a response which was able to effectively discriminate between differential types of bacterial infection via the characterised Drosophila systemic immunity pathways. Live-imaging studies also showed that the cellular immune response was functional within the Stage 15 embryo. Subsequently, immune competence was shown to arise at approximately mid-embryogenesis, under the control of 20-hydroxyecdysone (20-HE) signalling, as demonstrated by the partial rescue of AMP expression and bacterial clearance in early stage embryos upon 20-HE co-administration with infective agents. Further analysis of the global transcriptional response of the Drosophila embryo to infection and damage via microarray studies confirmed the immune potential of this system, but also permitted the identification of genes upregulated uniquely upon Gram-positive or Gram-negativ infection. Moreover, wounding via sterile laser ablation induced significant upregulation of a subset of AMP genes an a network of cuticular genes, providing an insight into the embryonic damage response. Parallel analysis of the hemocyte transcriptional profile upon infection and damage elucidated that these immune cells may play a role in the regulation of the immune response via 20-HE signalling and production of ROS, although this remains subject to further validation. As such, transcriptional profile analysis of the embryo has been successful in identifying candidate genes for further validation and study.

<p>The fruit fly <i>Drosophila melanogaster</i> has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. </p><p>We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the <i>Drosophila</i> immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G^-) bacteria and some Gram-positive (G⁺) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. </p><p>Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. </p><p>By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the <i>Drosophila</i> immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the <i>Drosophila</i> PRRs can recognize G^- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G⁺ bacteria such as <i>Micrococcus luteus</i> and <i>Bacillus megaterium </i>and fungal PAMPs from <i>Beauveria bassiana</i> and <i>Geotrichum candidum</i>. </p><p>This thesis also investigates the expression of the antimicrobial peptide genes, <i>Diptericin B</i> and <i>Attacin C</i>, and the putative intracellular antimicrobial peptide gene <i>Attacin D</i>, and explores a potential evolutionary link between them.</p>

The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum. This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.

All eukaryotic organisms have an innate immune system characterized by germ-line encoded receptors and effector molecules, which mediate detection and clearance of microbes such as bacteria, fungi, and parasites. VertebrateDrosophila as a genetically tractable organism with a This thesis concerns the peptidoglycan recognition protein (PGRP) gene family in the fruit fly. The family consists of thirteen genes, of which a few have been reported to be part of the signaling pathways that regulates immune Data presented show that the putative receptors have affinity for peptidoglycan, but not for lipopolysaccharide, or the fungal cell wall polymer beta-glucan. PGRP-SA, receptor of the Toll pathway, has a preference for In a search for novel PGRP receptors I found two PGRP proteins that instead displayed enzymatic activity towards peptidoglycan. They are of the N-actylmuramoyl L-alanine amidase type, which degrades peptidoglycan by splittingStaphylococcus aureus peptidoglycan looses its immune elicitor capacity. This is in contrast to lysozyme-degraded peptidoglycan, which isDrosophila PGRPs to be potential enzymes. PGRP-SB1 is the other enzymatic PGRP described within this thesis. It has a moreBacillus megaterium. In conclusion, receptor PGRP proteins binds bacterial peptidoglycan and triggers immune gene pathways and enzymatic PGRPs have the capacity to reduce the elicitor property of peptidoglycan.