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Differential activation of the NF-kappaB-like factors Relish and Dif in Drosophila melanogaster by fungi and gram-positive bacteria
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Dept. of Microbiology, Oregon State University, Corvallis.
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
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2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, Vol. 279, no 20, 21121-7 p.Article in journal (Refereed) Published
Abstract [en]

The current model of immune activation in Drosophila melanogaster suggests that fungi and Gram-positive (G+) bacteria activate the Toll/Dif pathway and that Gram-negative (G-) bacteria activate the Imd/Relish pathway. To test this model, we examined the response of Relish and Dif (Dorsal-related immunity factor) mutants to challenge by various fungi and G+ and G- bacteria. In Relish mutants, the Cecropin A gene was induced by the G+ bacteria Micrococcus luteus and Staphylococcus aureus, but not by other G+ or G- bacteria. This Relish-independent Cecropin A induction was blocked in Dif/Relish double mutant flies. Induction of the Cecropin A1 gene by M. luteus required Relish, whereas induction of the Cecropin A2 gene required Dif. Intact peptidoglycan (PG) was necessary for this differential induction of Cecropin A. PG extracted from M. luteus induced Cecropin A in Relish mutants, whereas PGs from the G+ bacteria Bacillus megaterium and Bacillus subtilis did not, suggesting that the Drosophila immune system can distinguish PGs from various G+ bacteria. Various fungi stimulated antimicrobial peptides through at least two different pathways requiring Relish and/or Dif. Induction of Attacin A by Geotrichum candidum required Relish, whereas activation by Beauvaria bassiana required Dif, suggesting that the Drosophila immune system can distinguish between at least these two fungi. We conclude that the Drosophila immune system is more complex than the current model. We propose a new model to account for this immune system complexity, incorporating distinct pattern recognition receptors of the Drosophila immune system, which can distinguish between various fungi and G+ bacteria, thereby leading to selective induction of antimicrobial peptides via differential activation of Relish and Dif.

Place, publisher, year, edition, pages
American Society for Biochemistry and Molecular Biology , 2004. Vol. 279, no 20, 21121-7 p.
National Category
Cell Biology
Research subject
URN: urn:nbn:se:su:diva-23158DOI: 10.1074/jbc.M313856200OAI: diva2:190443
Part of urn:nbn:se:su:diva-170Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2009-12-22Bibliographically approved
In thesis
1. Relish and the Regulation of Antimicrobial Peptides in Drosophila melanogaster
Open this publication in new window or tab >>Relish and the Regulation of Antimicrobial Peptides in Drosophila melanogaster
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2004. 49 p.
Drosophila immunity, NF-kappaB, Relish, antimicrobial peptides
National Category
Developmental Biology
urn:nbn:se:su:diva-170 (URN)91-7265-897-5 (ISBN)
Public defence
2004-06-02, hörsalen, Frescati backe, Svante Arrhenius väg 21 A, Stockholm, 13:00
Available from: 2004-05-13 Created: 2004-05-13 Last updated: 2011-03-28Bibliographically approved

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