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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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The effect of Edin on AMP production in vivo.Edin RNAi and overexpression flies (edin,RelE20) were crossed with C564-GAL4 flies or w1118 flies as a control, their offspring was infected with E. cloacae, total RNAs were extracted at indicated time points and qRT-PCR for the indicated genes was performed. (A) Expression of edin is knocked down in edin RNAi flies crossed to C564-GAL4 driver flies. (B) Edin overexpression flies express edin at a physiological level. Edin overexpression flies crossed with C564-GAL4 have slightly higher levels of edin compared to flies crossed with w1118. For (A–B) the data are pooled from 2 independent experiments, and n = 8 for each sample at each time point. (C–H) The effect of edin RNAi and overexpression on the production of Diptericin B (C), Cecropin A1 (D), Attacin B (E), Attacin A (F), Attacin C (G) and Drosocin (H). n = 4 for each sample at each time point. Error bars represent the standard deviation of each sample.
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pone-0037153-g005: The effect of Edin on AMP production in vivo.Edin RNAi and overexpression flies (edin,RelE20) were crossed with C564-GAL4 flies or w1118 flies as a control, their offspring was infected with E. cloacae, total RNAs were extracted at indicated time points and qRT-PCR for the indicated genes was performed. (A) Expression of edin is knocked down in edin RNAi flies crossed to C564-GAL4 driver flies. (B) Edin overexpression flies express edin at a physiological level. Edin overexpression flies crossed with C564-GAL4 have slightly higher levels of edin compared to flies crossed with w1118. For (A–B) the data are pooled from 2 independent experiments, and n = 8 for each sample at each time point. (C–H) The effect of edin RNAi and overexpression on the production of Diptericin B (C), Cecropin A1 (D), Attacin B (E), Attacin A (F), Attacin C (G) and Drosocin (H). n = 4 for each sample at each time point. Error bars represent the standard deviation of each sample.

Mentions: To test the role of Edin in Imd pathway regulation in vivo, we monitored the Imd pathway-mediated AMP gene expression levels with qRT-PCR in edin RNAi flies and in edin overexpression flies we created. The overexpression flies were created by microinjecting the pUAST-edin construct into RelE20 mutant embryos. To analyze Imd pathway activity, edin RNAi (VDRC #14289) and UAS-edin,RelE20 flies were crossed with the C564-GAL4 driver that targets transgene expression to the fat body in addition to some other organs [22]. The Imd pathway was then activated in week-old offspring by septic injury with E. cloacae. Flies crossed with w1118 flies were used as controls. As shown in Figure 5A, in vivo RNAi of edin using the C564-GAL4 driver strongly suppresses edin expression in whole flies, indicating that the UAS-RNAi construct is effective. UAS-edin,RelE20 flies crossed with the C564-GAL4 driver showed expression levels comparable to the E. cloacae infected control flies (Figure 5B).


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)

The effect of Edin on AMP production in vivo.Edin RNAi and overexpression flies (edin,RelE20) were crossed with C564-GAL4 flies or w1118 flies as a control, their offspring was infected with E. cloacae, total RNAs were extracted at indicated time points and qRT-PCR for the indicated genes was performed. (A) Expression of edin is knocked down in edin RNAi flies crossed to C564-GAL4 driver flies. (B) Edin overexpression flies express edin at a physiological level. Edin overexpression flies crossed with C564-GAL4 have slightly higher levels of edin compared to flies crossed with w1118. For (A–B) the data are pooled from 2 independent experiments, and n = 8 for each sample at each time point. (C–H) The effect of edin RNAi and overexpression on the production of Diptericin B (C), Cecropin A1 (D), Attacin B (E), Attacin A (F), Attacin C (G) and Drosocin (H). n = 4 for each sample at each time point. Error bars represent the standard deviation of each sample.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037153-g005: The effect of Edin on AMP production in vivo.Edin RNAi and overexpression flies (edin,RelE20) were crossed with C564-GAL4 flies or w1118 flies as a control, their offspring was infected with E. cloacae, total RNAs were extracted at indicated time points and qRT-PCR for the indicated genes was performed. (A) Expression of edin is knocked down in edin RNAi flies crossed to C564-GAL4 driver flies. (B) Edin overexpression flies express edin at a physiological level. Edin overexpression flies crossed with C564-GAL4 have slightly higher levels of edin compared to flies crossed with w1118. For (A–B) the data are pooled from 2 independent experiments, and n = 8 for each sample at each time point. (C–H) The effect of edin RNAi and overexpression on the production of Diptericin B (C), Cecropin A1 (D), Attacin B (E), Attacin A (F), Attacin C (G) and Drosocin (H). n = 4 for each sample at each time point. Error bars represent the standard deviation of each sample.
Mentions: To test the role of Edin in Imd pathway regulation in vivo, we monitored the Imd pathway-mediated AMP gene expression levels with qRT-PCR in edin RNAi flies and in edin overexpression flies we created. The overexpression flies were created by microinjecting the pUAST-edin construct into RelE20 mutant embryos. To analyze Imd pathway activity, edin RNAi (VDRC #14289) and UAS-edin,RelE20 flies were crossed with the C564-GAL4 driver that targets transgene expression to the fat body in addition to some other organs [22]. The Imd pathway was then activated in week-old offspring by septic injury with E. cloacae. Flies crossed with w1118 flies were used as controls. As shown in Figure 5A, in vivo RNAi of edin using the C564-GAL4 driver strongly suppresses edin expression in whole flies, indicating that the UAS-RNAi construct is effective. UAS-edin,RelE20 flies crossed with the C564-GAL4 driver showed expression levels comparable to the E. cloacae infected control flies (Figure 5B).

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