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Functional characterization of the infection-inducible peptide Edin in Drosophila melanogaster.

Vanha-Aho LM, Kleino A, Kaustio M, Ulvila J, Wilke B, Hultmark D, Valanne S, Rämet M - PLoS ONE (2012)

Bottom Line: In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved.In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo.Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech and Institute of Biomedical Technology, University of Tampere, Tampere, Finland.

ABSTRACT
Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response.

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Overexpressing Edin has no effect on fly survival after Gram-positive or Gram-negative bacterial challenge in vivo.(A) Overexpressing edin does not negatively affect lifespan. UAS-edin overexpression flies were crossed with Actin5C-GAL4 driver lines and the lifespan of their offspring was followed. w1118 crossed with RelE20 mutants and Act5C-GAL4 crossed with RelE20 were used as controls. The data represent one experiment, n = 100 for each cross. (B) Survival is not negatively affected in UAS-edin overexpressing flies. Equal amounts of edin,RelE20/Act5C-GAL4 and edin,RelE20/CyO genotypes were obtained from the crosses. (C–H) Flies were pricked with the indicated microbe and survival was followed. (C–E) Overexpressing edin in the RelE20 background does not protect the flies from L. monocytogenes (C), E. cloacae (D) or E. faecalis (E) infection. In C–E RelE20 crossed with edin,RelE20 and C564;RelE20 crossed with RelE20 were used as controls. (F–H) Overexpressing edin in a heterozygous w1118 background does not protect the flies from L. monocytogenes (F), E. cloacae (G) or E. faecalis (H) infection. Edin overexpression flies were pricked with E. faecalis, E. faecalis or L. monocytogenes. C564-GAL4 flies crossed with w1118 and UAS-edin,RelE20 crossed with w1118 flies were used as controls. Data are pooled from 2–3 experiments which showed similar trends, for each cross (D–J) n = 34–118.
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pone-0037153-g007: Overexpressing Edin has no effect on fly survival after Gram-positive or Gram-negative bacterial challenge in vivo.(A) Overexpressing edin does not negatively affect lifespan. UAS-edin overexpression flies were crossed with Actin5C-GAL4 driver lines and the lifespan of their offspring was followed. w1118 crossed with RelE20 mutants and Act5C-GAL4 crossed with RelE20 were used as controls. The data represent one experiment, n = 100 for each cross. (B) Survival is not negatively affected in UAS-edin overexpressing flies. Equal amounts of edin,RelE20/Act5C-GAL4 and edin,RelE20/CyO genotypes were obtained from the crosses. (C–H) Flies were pricked with the indicated microbe and survival was followed. (C–E) Overexpressing edin in the RelE20 background does not protect the flies from L. monocytogenes (C), E. cloacae (D) or E. faecalis (E) infection. In C–E RelE20 crossed with edin,RelE20 and C564;RelE20 crossed with RelE20 were used as controls. (F–H) Overexpressing edin in a heterozygous w1118 background does not protect the flies from L. monocytogenes (F), E. cloacae (G) or E. faecalis (H) infection. Edin overexpression flies were pricked with E. faecalis, E. faecalis or L. monocytogenes. C564-GAL4 flies crossed with w1118 and UAS-edin,RelE20 crossed with w1118 flies were used as controls. Data are pooled from 2–3 experiments which showed similar trends, for each cross (D–J) n = 34–118.

Mentions: To test the antimicrobial properties of Edin in a more physiological context, the effect of edin overexpression on the survival of flies after bacterial infections was analyzed. First, to test whether overexpressing edin affects survival or lifespan, the UAS-edin,RelE20 overexpression line was crossed with the Act5C-GAL4/CyO driver line and the lifespan of the offspring was monitored. As shown in Figure 7A, overexpression of edin did not affect the lifespan of the flies and was comparable to that of the control flies. Furthermore, edin expression did not compromise the development of flies since equal amounts of UAS-edin,RelE20/ActGAL4 and UAS-edin,RelE20/CyO flies were obtained from the crosses (Figure 7B). Similar results were obtained when edin overexpression flies where crossed with either the C564-GAL4 driver line or the ubiquitous daughterless-GAL4 driver line (data not shown).


Functional characterization of the infection-inducible peptide Edin in Drosophila melanogaster.

Vanha-Aho LM, Kleino A, Kaustio M, Ulvila J, Wilke B, Hultmark D, Valanne S, Rämet M - PLoS ONE (2012)

Overexpressing Edin has no effect on fly survival after Gram-positive or Gram-negative bacterial challenge in vivo.(A) Overexpressing edin does not negatively affect lifespan. UAS-edin overexpression flies were crossed with Actin5C-GAL4 driver lines and the lifespan of their offspring was followed. w1118 crossed with RelE20 mutants and Act5C-GAL4 crossed with RelE20 were used as controls. The data represent one experiment, n = 100 for each cross. (B) Survival is not negatively affected in UAS-edin overexpressing flies. Equal amounts of edin,RelE20/Act5C-GAL4 and edin,RelE20/CyO genotypes were obtained from the crosses. (C–H) Flies were pricked with the indicated microbe and survival was followed. (C–E) Overexpressing edin in the RelE20 background does not protect the flies from L. monocytogenes (C), E. cloacae (D) or E. faecalis (E) infection. In C–E RelE20 crossed with edin,RelE20 and C564;RelE20 crossed with RelE20 were used as controls. (F–H) Overexpressing edin in a heterozygous w1118 background does not protect the flies from L. monocytogenes (F), E. cloacae (G) or E. faecalis (H) infection. Edin overexpression flies were pricked with E. faecalis, E. faecalis or L. monocytogenes. C564-GAL4 flies crossed with w1118 and UAS-edin,RelE20 crossed with w1118 flies were used as controls. Data are pooled from 2–3 experiments which showed similar trends, for each cross (D–J) n = 34–118.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351453&req=5

pone-0037153-g007: Overexpressing Edin has no effect on fly survival after Gram-positive or Gram-negative bacterial challenge in vivo.(A) Overexpressing edin does not negatively affect lifespan. UAS-edin overexpression flies were crossed with Actin5C-GAL4 driver lines and the lifespan of their offspring was followed. w1118 crossed with RelE20 mutants and Act5C-GAL4 crossed with RelE20 were used as controls. The data represent one experiment, n = 100 for each cross. (B) Survival is not negatively affected in UAS-edin overexpressing flies. Equal amounts of edin,RelE20/Act5C-GAL4 and edin,RelE20/CyO genotypes were obtained from the crosses. (C–H) Flies were pricked with the indicated microbe and survival was followed. (C–E) Overexpressing edin in the RelE20 background does not protect the flies from L. monocytogenes (C), E. cloacae (D) or E. faecalis (E) infection. In C–E RelE20 crossed with edin,RelE20 and C564;RelE20 crossed with RelE20 were used as controls. (F–H) Overexpressing edin in a heterozygous w1118 background does not protect the flies from L. monocytogenes (F), E. cloacae (G) or E. faecalis (H) infection. Edin overexpression flies were pricked with E. faecalis, E. faecalis or L. monocytogenes. C564-GAL4 flies crossed with w1118 and UAS-edin,RelE20 crossed with w1118 flies were used as controls. Data are pooled from 2–3 experiments which showed similar trends, for each cross (D–J) n = 34–118.
Mentions: To test the antimicrobial properties of Edin in a more physiological context, the effect of edin overexpression on the survival of flies after bacterial infections was analyzed. First, to test whether overexpressing edin affects survival or lifespan, the UAS-edin,RelE20 overexpression line was crossed with the Act5C-GAL4/CyO driver line and the lifespan of the offspring was monitored. As shown in Figure 7A, overexpression of edin did not affect the lifespan of the flies and was comparable to that of the control flies. Furthermore, edin expression did not compromise the development of flies since equal amounts of UAS-edin,RelE20/ActGAL4 and UAS-edin,RelE20/CyO flies were obtained from the crosses (Figure 7B). Similar results were obtained when edin overexpression flies where crossed with either the C564-GAL4 driver line or the ubiquitous daughterless-GAL4 driver line (data not shown).

Bottom Line: In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved.In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo.Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo.

View Article: PubMed Central - PubMed

Affiliation: BioMediTech and Institute of Biomedical Technology, University of Tampere, Tampere, Finland.

ABSTRACT
Drosophila is a well-established model organism for studying innate immunity because of its high resistance against microbial infections and lack of adaptive immunity. In addition, the immune signaling cascades found in Drosophila are evolutionarily conserved. Upon infection, activation of the immune signaling pathways, Toll and Imd, leads to the expression of multiple immune response genes, such as the antimicrobial peptides (AMPs). Previously, we identified an uncharacterized gene edin among the genes, which were strongly induced upon stimulation with Escherichia coli in Drosophila S2 cells. Edin has been associated with resistance against Listeria monocytogenes, but its role in Drosophila immunity remains elusive. In this study, we examined the role of Edin in the immune response of Drosophila both in vitro and in vivo. We report that edin expression is dependent on the Imd-pathway NF-κB transcription factor Relish and that it is expressed upon infection both in vitro and in vivo. Edin encodes a pro-protein, which is further processed in S2 cells. In our experiments, Edin did not bind microbes, nor did it possess antimicrobial activity to tested microbial strains in vitro or in vivo. Furthermore, edin RNAi did not significantly affect the expression of AMPs in vitro or in vivo. However, edin RNAi flies showed modestly impaired resistance to E. faecalis infection. We conclude that Edin has no potent antimicrobial properties but it appears to be important for E. faecalis infection via an uncharacterized mechanism. Further studies are still required to elucidate the exact role of Edin in the Drosophila immune response.

Show MeSH
Related in: MedlinePlus