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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 does not affect the ability of S2 cells to bind microbes.(A–B) The effect of edin RNAi on the binding of E. coli and S. aureus in Drosophila S2 cells. Drosophila S2 cells were soaked for three days in dsRNAs and thereafter exposed to bacteria at +4°C. GFP dsRNA was used as a negative and eater dsRNA as a positive control. (C–D) The effect of edin overexpression on the binding of E. coli and S. aureus. S2 cells were transiently transfected with a pMT construct expressing edin and endogenous edin expression was knocked down with dsRNA treatments. The ability of S2 cells to bind heat-killed E. coli (A, C) or S. aureus (B, D) was measured using flow cytometry.
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pone-0037153-g002: Edin does not affect the ability of S2 cells to bind microbes.(A–B) The effect of edin RNAi on the binding of E. coli and S. aureus in Drosophila S2 cells. Drosophila S2 cells were soaked for three days in dsRNAs and thereafter exposed to bacteria at +4°C. GFP dsRNA was used as a negative and eater dsRNA as a positive control. (C–D) The effect of edin overexpression on the binding of E. coli and S. aureus. S2 cells were transiently transfected with a pMT construct expressing edin and endogenous edin expression was knocked down with dsRNA treatments. The ability of S2 cells to bind heat-killed E. coli (A, C) or S. aureus (B, D) was measured using flow cytometry.

Mentions: The phagocytosis of invading microbes is an essential component of Drosophila immunity [18], [19]. To this end we tested whether Edin has a role in bacterial binding or opsonization. Plasmatocyte-like S2 cells that are capable of binding and phagocytosing microbes [20] were treated with edin dsRNA and the ability of the cells to bind heat-killed, fluorescently labeled E. coli and Staphylococcus aureus was analyzed with flow cytometry. As a positive control, we used a dsRNA treatment targeting eater, which codes for an important phagocytic receptor for bacteria both in S2 cells and in Drosophila in vivo[18], [19], [21]. GFP dsRNA was used as a negative control. Edin RNAi did not affect the ability of S2 cells to bind E. coli (Figure 2A). Likewise, edin dsRNA treatments did not compromise the ability of S2 cells to bind S. aureus (Figure 2B) but rather seemed to modestly enhance the binding activity of S2 cells.


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 does not affect the ability of S2 cells to bind microbes.(A–B) The effect of edin RNAi on the binding of E. coli and S. aureus in Drosophila S2 cells. Drosophila S2 cells were soaked for three days in dsRNAs and thereafter exposed to bacteria at +4°C. GFP dsRNA was used as a negative and eater dsRNA as a positive control. (C–D) The effect of edin overexpression on the binding of E. coli and S. aureus. S2 cells were transiently transfected with a pMT construct expressing edin and endogenous edin expression was knocked down with dsRNA treatments. The ability of S2 cells to bind heat-killed E. coli (A, C) or S. aureus (B, D) was measured using flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037153-g002: Edin does not affect the ability of S2 cells to bind microbes.(A–B) The effect of edin RNAi on the binding of E. coli and S. aureus in Drosophila S2 cells. Drosophila S2 cells were soaked for three days in dsRNAs and thereafter exposed to bacteria at +4°C. GFP dsRNA was used as a negative and eater dsRNA as a positive control. (C–D) The effect of edin overexpression on the binding of E. coli and S. aureus. S2 cells were transiently transfected with a pMT construct expressing edin and endogenous edin expression was knocked down with dsRNA treatments. The ability of S2 cells to bind heat-killed E. coli (A, C) or S. aureus (B, D) was measured using flow cytometry.
Mentions: The phagocytosis of invading microbes is an essential component of Drosophila immunity [18], [19]. To this end we tested whether Edin has a role in bacterial binding or opsonization. Plasmatocyte-like S2 cells that are capable of binding and phagocytosing microbes [20] were treated with edin dsRNA and the ability of the cells to bind heat-killed, fluorescently labeled E. coli and Staphylococcus aureus was analyzed with flow cytometry. As a positive control, we used a dsRNA treatment targeting eater, which codes for an important phagocytic receptor for bacteria both in S2 cells and in Drosophila in vivo[18], [19], [21]. GFP dsRNA was used as a negative control. Edin RNAi did not affect the ability of S2 cells to bind E. coli (Figure 2A). Likewise, edin dsRNA treatments did not compromise the ability of S2 cells to bind S. aureus (Figure 2B) but rather seemed to modestly enhance the binding activity of S2 cells.

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