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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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Effect of edin RNAi on Drosophila immune signaling in vitro.A) Edin RNAi is effective in S2 cells. S2 cells were treated with GFP and edin dsRNA and the cells were induced by adding heat-killed E. coli. Relative expression levels of edin were analyzed from total RNAs with qRT-PCR. n = 4 for each sample. (B) Edin expression is not required for the Imd pathway signaling in vitro. S2 cells were transfected with an Attacin A-luciferase reporter together with GFP (negative control), Relish (positive control) and edin dsRNAs. The Imd pathway was activated by adding heat-killed E. coli to the cell culture medium and samples were collected at indicated time points. Edin RNAi causes a 30% decrease in the Imd pathway activity at the 24 h time point. The data for the 0 h and 24 h time points are pooled from 5 indepent experiments (n = 17 per sample). For 1 h, 4 h and 8 h time points n = 4 per sample. (C) Edin RNAi does not decrease the Imd pathway activity when the pathway is induced with S. marcescens, E. cloacae, peptidoglycan or PGRP-LC. S2 cells were transfected with an AttA-luciferase reporter and edin dsRNA and the Imd pathway was activated with S. marcescens (S.m.), E. cloacae (E.cl.), peptidoglycan (PGN) or a pMT[PGRP-LC] construct. CuSO4 was used to induce the expression of PGRP-LC. GFP and Relish dsRNAs were used as negative and positive controls, respectively. Unind. = no induction. The data for S.m., E.cl. and PGN are pooled from 3 independent experiments (n = 12 per sample). For PGRP-LC, n = 3 per sample. (D) Edin RNAi does not affect the Toll pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin and MyD88 (positive control) dsRNAs. A constitutively active form of the Toll receptor, Toll10B, was used to activate the pathway. The data are pooled from 3 independent experiments, n = 10 for each sample. (E) Edin has no effect on the Spätzle-induced Toll-pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin, MyD88 (control) and Cactus (control) dsRNAs. The Toll pathway was activated with the cleaved, active Spätzle ligand (SpzC106). n = 4 for each sample. (F) Edin RNAi has no effect on the JAK/STAT pathway. S2 cells were transfected with a Turandot M-reporter and GFP, STAT (positive control) and edin dsRNAs. The JAK/STAT pathway was activated by overexpressing HopTum-l. n = 4 for each sample.
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pone-0037153-g004: Effect of edin RNAi on Drosophila immune signaling in vitro.A) Edin RNAi is effective in S2 cells. S2 cells were treated with GFP and edin dsRNA and the cells were induced by adding heat-killed E. coli. Relative expression levels of edin were analyzed from total RNAs with qRT-PCR. n = 4 for each sample. (B) Edin expression is not required for the Imd pathway signaling in vitro. S2 cells were transfected with an Attacin A-luciferase reporter together with GFP (negative control), Relish (positive control) and edin dsRNAs. The Imd pathway was activated by adding heat-killed E. coli to the cell culture medium and samples were collected at indicated time points. Edin RNAi causes a 30% decrease in the Imd pathway activity at the 24 h time point. The data for the 0 h and 24 h time points are pooled from 5 indepent experiments (n = 17 per sample). For 1 h, 4 h and 8 h time points n = 4 per sample. (C) Edin RNAi does not decrease the Imd pathway activity when the pathway is induced with S. marcescens, E. cloacae, peptidoglycan or PGRP-LC. S2 cells were transfected with an AttA-luciferase reporter and edin dsRNA and the Imd pathway was activated with S. marcescens (S.m.), E. cloacae (E.cl.), peptidoglycan (PGN) or a pMT[PGRP-LC] construct. CuSO4 was used to induce the expression of PGRP-LC. GFP and Relish dsRNAs were used as negative and positive controls, respectively. Unind. = no induction. The data for S.m., E.cl. and PGN are pooled from 3 independent experiments (n = 12 per sample). For PGRP-LC, n = 3 per sample. (D) Edin RNAi does not affect the Toll pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin and MyD88 (positive control) dsRNAs. A constitutively active form of the Toll receptor, Toll10B, was used to activate the pathway. The data are pooled from 3 independent experiments, n = 10 for each sample. (E) Edin has no effect on the Spätzle-induced Toll-pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin, MyD88 (control) and Cactus (control) dsRNAs. The Toll pathway was activated with the cleaved, active Spätzle ligand (SpzC106). n = 4 for each sample. (F) Edin RNAi has no effect on the JAK/STAT pathway. S2 cells were transfected with a Turandot M-reporter and GFP, STAT (positive control) and edin dsRNAs. The JAK/STAT pathway was activated by overexpressing HopTum-l. n = 4 for each sample.

Mentions: Next, we investigated whether Edin is involved in modulating the activity of Drosophila innate immune signaling cascades. S2 cells were transfected with luciferase-reporter constructs together with edin dsRNA as well as with negative and positive control dsRNAs, and the luciferase activities of the cell lysates were analyzed. Transfection efficacy and cell viability were assessed with an Actin 5C-β-galactosidase reporter. GFP dsRNA was used as a negative control in all assays. First, we tested the effectiveness of edin RNAi in vitro by treating S2 cells with GFP or edin dsRNAs, and analyzing the relative expression levels of edin. As shown in Figure 4A, edin RNAi abolishes the endogenous edin expression.


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)

Effect of edin RNAi on Drosophila immune signaling in vitro.A) Edin RNAi is effective in S2 cells. S2 cells were treated with GFP and edin dsRNA and the cells were induced by adding heat-killed E. coli. Relative expression levels of edin were analyzed from total RNAs with qRT-PCR. n = 4 for each sample. (B) Edin expression is not required for the Imd pathway signaling in vitro. S2 cells were transfected with an Attacin A-luciferase reporter together with GFP (negative control), Relish (positive control) and edin dsRNAs. The Imd pathway was activated by adding heat-killed E. coli to the cell culture medium and samples were collected at indicated time points. Edin RNAi causes a 30% decrease in the Imd pathway activity at the 24 h time point. The data for the 0 h and 24 h time points are pooled from 5 indepent experiments (n = 17 per sample). For 1 h, 4 h and 8 h time points n = 4 per sample. (C) Edin RNAi does not decrease the Imd pathway activity when the pathway is induced with S. marcescens, E. cloacae, peptidoglycan or PGRP-LC. S2 cells were transfected with an AttA-luciferase reporter and edin dsRNA and the Imd pathway was activated with S. marcescens (S.m.), E. cloacae (E.cl.), peptidoglycan (PGN) or a pMT[PGRP-LC] construct. CuSO4 was used to induce the expression of PGRP-LC. GFP and Relish dsRNAs were used as negative and positive controls, respectively. Unind. = no induction. The data for S.m., E.cl. and PGN are pooled from 3 independent experiments (n = 12 per sample). For PGRP-LC, n = 3 per sample. (D) Edin RNAi does not affect the Toll pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin and MyD88 (positive control) dsRNAs. A constitutively active form of the Toll receptor, Toll10B, was used to activate the pathway. The data are pooled from 3 independent experiments, n = 10 for each sample. (E) Edin has no effect on the Spätzle-induced Toll-pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin, MyD88 (control) and Cactus (control) dsRNAs. The Toll pathway was activated with the cleaved, active Spätzle ligand (SpzC106). n = 4 for each sample. (F) Edin RNAi has no effect on the JAK/STAT pathway. S2 cells were transfected with a Turandot M-reporter and GFP, STAT (positive control) and edin dsRNAs. The JAK/STAT pathway was activated by overexpressing HopTum-l. n = 4 for each sample.
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pone-0037153-g004: Effect of edin RNAi on Drosophila immune signaling in vitro.A) Edin RNAi is effective in S2 cells. S2 cells were treated with GFP and edin dsRNA and the cells were induced by adding heat-killed E. coli. Relative expression levels of edin were analyzed from total RNAs with qRT-PCR. n = 4 for each sample. (B) Edin expression is not required for the Imd pathway signaling in vitro. S2 cells were transfected with an Attacin A-luciferase reporter together with GFP (negative control), Relish (positive control) and edin dsRNAs. The Imd pathway was activated by adding heat-killed E. coli to the cell culture medium and samples were collected at indicated time points. Edin RNAi causes a 30% decrease in the Imd pathway activity at the 24 h time point. The data for the 0 h and 24 h time points are pooled from 5 indepent experiments (n = 17 per sample). For 1 h, 4 h and 8 h time points n = 4 per sample. (C) Edin RNAi does not decrease the Imd pathway activity when the pathway is induced with S. marcescens, E. cloacae, peptidoglycan or PGRP-LC. S2 cells were transfected with an AttA-luciferase reporter and edin dsRNA and the Imd pathway was activated with S. marcescens (S.m.), E. cloacae (E.cl.), peptidoglycan (PGN) or a pMT[PGRP-LC] construct. CuSO4 was used to induce the expression of PGRP-LC. GFP and Relish dsRNAs were used as negative and positive controls, respectively. Unind. = no induction. The data for S.m., E.cl. and PGN are pooled from 3 independent experiments (n = 12 per sample). For PGRP-LC, n = 3 per sample. (D) Edin RNAi does not affect the Toll pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin and MyD88 (positive control) dsRNAs. A constitutively active form of the Toll receptor, Toll10B, was used to activate the pathway. The data are pooled from 3 independent experiments, n = 10 for each sample. (E) Edin has no effect on the Spätzle-induced Toll-pathway activity. S2 cells were transfected with a Drosomycin-luciferase reporter together with GFP, edin, MyD88 (control) and Cactus (control) dsRNAs. The Toll pathway was activated with the cleaved, active Spätzle ligand (SpzC106). n = 4 for each sample. (F) Edin RNAi has no effect on the JAK/STAT pathway. S2 cells were transfected with a Turandot M-reporter and GFP, STAT (positive control) and edin dsRNAs. The JAK/STAT pathway was activated by overexpressing HopTum-l. n = 4 for each sample.
Mentions: Next, we investigated whether Edin is involved in modulating the activity of Drosophila innate immune signaling cascades. S2 cells were transfected with luciferase-reporter constructs together with edin dsRNA as well as with negative and positive control dsRNAs, and the luciferase activities of the cell lysates were analyzed. Transfection efficacy and cell viability were assessed with an Actin 5C-β-galactosidase reporter. GFP dsRNA was used as a negative control in all assays. First, we tested the effectiveness of edin RNAi in vitro by treating S2 cells with GFP or edin dsRNAs, and analyzing the relative expression levels of edin. As shown in Figure 4A, edin RNAi abolishes the endogenous edin expression.

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