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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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Edin is a Relish-dependently synthesized peptide, which is secreted from S2 cells.(A–B) Edin contains a signal sequence and is secreted from S2 cells. (A) Edin sequences are aligned from 12 Drosophila species and three other dipterans. Diptericin B and Attacin A from D. melanogaster are also included in the alignment. The predicted signal peptidase cleavage sites [31] are marked. The sequences from the 12 Drosophila species are all from Clark et al. 2007 [32], except the D. mojavensis sequence which is derived from an EST sequence (EB600147). Modified gene models without introns were used for D. yakuba and D. willistoni. The Lucilia sericata sequence is derived from a single EST (FG360503). Three Stomoxys calcitrans ESTs (DN952426, DN952940, EZ048833) and one Glossina morsitans EST (AF368915) appear to contain overlapping sequence from the same gene. The Musca domestica sequence is an isoform represented by one EST (ES608713). (B) The signal sequence of Edin is cleaved before the peptide is secreted to the cell culture medium. S2 cells were transfected with a pMT-edin-V5 construct and the cell culture medium and cell lysates were analyzed with western blotting. Both full-length and cleaved forms were observed in the lysates while only the cleaved form was present in the medium. The V5 tag is located at the C-terminus of Edin. The blot represents 4 independent samples from which both cell lysates and culture medium were analyzed. (C) Edin is induced upon Enterobacter cloacae infection in Canton S flies but not in RelE20 flies. Canton S flies and RelE20-mutant flies were pricked with E. cloacae and total RNAs were extracted at the indicated time points. RT-PCR was performed and samples were electrophoresed on an agarose gel. Actin5C was used as a loading control and Attacin A as a positive control.
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pone-0037153-g001: Edin is a Relish-dependently synthesized peptide, which is secreted from S2 cells.(A–B) Edin contains a signal sequence and is secreted from S2 cells. (A) Edin sequences are aligned from 12 Drosophila species and three other dipterans. Diptericin B and Attacin A from D. melanogaster are also included in the alignment. The predicted signal peptidase cleavage sites [31] are marked. The sequences from the 12 Drosophila species are all from Clark et al. 2007 [32], except the D. mojavensis sequence which is derived from an EST sequence (EB600147). Modified gene models without introns were used for D. yakuba and D. willistoni. The Lucilia sericata sequence is derived from a single EST (FG360503). Three Stomoxys calcitrans ESTs (DN952426, DN952940, EZ048833) and one Glossina morsitans EST (AF368915) appear to contain overlapping sequence from the same gene. The Musca domestica sequence is an isoform represented by one EST (ES608713). (B) The signal sequence of Edin is cleaved before the peptide is secreted to the cell culture medium. S2 cells were transfected with a pMT-edin-V5 construct and the cell culture medium and cell lysates were analyzed with western blotting. Both full-length and cleaved forms were observed in the lysates while only the cleaved form was present in the medium. The V5 tag is located at the C-terminus of Edin. The blot represents 4 independent samples from which both cell lysates and culture medium were analyzed. (C) Edin is induced upon Enterobacter cloacae infection in Canton S flies but not in RelE20 flies. Canton S flies and RelE20-mutant flies were pricked with E. cloacae and total RNAs were extracted at the indicated time points. RT-PCR was performed and samples were electrophoresed on an agarose gel. Actin5C was used as a loading control and Attacin A as a positive control.

Mentions: The edin gene encodes a short peptide of 115 amino acids including an N-terminal signal sequence (amino acids 1–22) (Figure 1A). The predicted signal peptidase cleavage site is supported by proteomic data from Verleyen et al. [16], who identified the predicted amino terminal of the mature protein in peptide fragments from hemolymph. Likely orthologs of the edin gene can be found in other brachyrecan flies, including all sequenced Drosophila species, but not in other insects (Figure 1A). For Musca domestica, three isoforms are represented in the EST databases (not shown). A tendency for pseudogenisation of the edin genes can be noted, as stop codons are present in the D. yakuba and D. mojavensis homologs. For the latter, an apparently functional allele is represented by an EST sequence (Figure 1A). A stop codon interrupts the open reading frame in the EST from Lucilia sericata, but this could be a sequencing error.


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)

Edin is a Relish-dependently synthesized peptide, which is secreted from S2 cells.(A–B) Edin contains a signal sequence and is secreted from S2 cells. (A) Edin sequences are aligned from 12 Drosophila species and three other dipterans. Diptericin B and Attacin A from D. melanogaster are also included in the alignment. The predicted signal peptidase cleavage sites [31] are marked. The sequences from the 12 Drosophila species are all from Clark et al. 2007 [32], except the D. mojavensis sequence which is derived from an EST sequence (EB600147). Modified gene models without introns were used for D. yakuba and D. willistoni. The Lucilia sericata sequence is derived from a single EST (FG360503). Three Stomoxys calcitrans ESTs (DN952426, DN952940, EZ048833) and one Glossina morsitans EST (AF368915) appear to contain overlapping sequence from the same gene. The Musca domestica sequence is an isoform represented by one EST (ES608713). (B) The signal sequence of Edin is cleaved before the peptide is secreted to the cell culture medium. S2 cells were transfected with a pMT-edin-V5 construct and the cell culture medium and cell lysates were analyzed with western blotting. Both full-length and cleaved forms were observed in the lysates while only the cleaved form was present in the medium. The V5 tag is located at the C-terminus of Edin. The blot represents 4 independent samples from which both cell lysates and culture medium were analyzed. (C) Edin is induced upon Enterobacter cloacae infection in Canton S flies but not in RelE20 flies. Canton S flies and RelE20-mutant flies were pricked with E. cloacae and total RNAs were extracted at the indicated time points. RT-PCR was performed and samples were electrophoresed on an agarose gel. Actin5C was used as a loading control and Attacin A as a positive control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037153-g001: Edin is a Relish-dependently synthesized peptide, which is secreted from S2 cells.(A–B) Edin contains a signal sequence and is secreted from S2 cells. (A) Edin sequences are aligned from 12 Drosophila species and three other dipterans. Diptericin B and Attacin A from D. melanogaster are also included in the alignment. The predicted signal peptidase cleavage sites [31] are marked. The sequences from the 12 Drosophila species are all from Clark et al. 2007 [32], except the D. mojavensis sequence which is derived from an EST sequence (EB600147). Modified gene models without introns were used for D. yakuba and D. willistoni. The Lucilia sericata sequence is derived from a single EST (FG360503). Three Stomoxys calcitrans ESTs (DN952426, DN952940, EZ048833) and one Glossina morsitans EST (AF368915) appear to contain overlapping sequence from the same gene. The Musca domestica sequence is an isoform represented by one EST (ES608713). (B) The signal sequence of Edin is cleaved before the peptide is secreted to the cell culture medium. S2 cells were transfected with a pMT-edin-V5 construct and the cell culture medium and cell lysates were analyzed with western blotting. Both full-length and cleaved forms were observed in the lysates while only the cleaved form was present in the medium. The V5 tag is located at the C-terminus of Edin. The blot represents 4 independent samples from which both cell lysates and culture medium were analyzed. (C) Edin is induced upon Enterobacter cloacae infection in Canton S flies but not in RelE20 flies. Canton S flies and RelE20-mutant flies were pricked with E. cloacae and total RNAs were extracted at the indicated time points. RT-PCR was performed and samples were electrophoresed on an agarose gel. Actin5C was used as a loading control and Attacin A as a positive control.
Mentions: The edin gene encodes a short peptide of 115 amino acids including an N-terminal signal sequence (amino acids 1–22) (Figure 1A). The predicted signal peptidase cleavage site is supported by proteomic data from Verleyen et al. [16], who identified the predicted amino terminal of the mature protein in peptide fragments from hemolymph. Likely orthologs of the edin gene can be found in other brachyrecan flies, including all sequenced Drosophila species, but not in other insects (Figure 1A). For Musca domestica, three isoforms are represented in the EST databases (not shown). A tendency for pseudogenisation of the edin genes can be noted, as stop codons are present in the D. yakuba and D. mojavensis homologs. For the latter, an apparently functional allele is represented by an EST sequence (Figure 1A). A stop codon interrupts the open reading frame in the EST from Lucilia sericata, but this could be a sequencing error.

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