Relish and the Regulation of Antimicrobial Peptides in Drosophila melanogaster
Sammanfattning: 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-?B family.We have generated loss-of-function mutants of the NF-?B-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 ?B-like sites in the promoter region.By studying the immune response of the Relish mutant flies in combination with mutants for another NF-?B-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.
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