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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 has no broad antimicrobial properties against Gram positive or Gram negative bacteria in vitro.(A–B) Edin does no limit the growth of E. coli or S. aureus in S2 cell culture medium. S2 cells were transfected with a copper-inducible pMT-edin-V5 or an empty pMT vector, and the abilities of E. coli and S. aureus to proliferate in these mediums were analyzed. (C–G) Synthetic forms of Edin do not limit the growth of E. coli (C), E. cloacae (D), L. monocytogenes (E), E. faecalis (F) or S. aureus (G). Both N-terminal and C-terminal forms of Edin were tested. Bacteria were cultured to an OD600 nm of 0.33, incubated with synthetic Edin and the ability of the bacteria to grow was analyzed. Cecropin A and Lysozyme were used as positive controls for Gram-negative and Gram-positive bacteria, respectively. Left column, N-terminal Edin; right column, C-terminal Edin.
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pone-0037153-g006: Edin has no broad antimicrobial properties against Gram positive or Gram negative bacteria in vitro.(A–B) Edin does no limit the growth of E. coli or S. aureus in S2 cell culture medium. S2 cells were transfected with a copper-inducible pMT-edin-V5 or an empty pMT vector, and the abilities of E. coli and S. aureus to proliferate in these mediums were analyzed. (C–G) Synthetic forms of Edin do not limit the growth of E. coli (C), E. cloacae (D), L. monocytogenes (E), E. faecalis (F) or S. aureus (G). Both N-terminal and C-terminal forms of Edin were tested. Bacteria were cultured to an OD600 nm of 0.33, incubated with synthetic Edin and the ability of the bacteria to grow was analyzed. Cecropin A and Lysozyme were used as positive controls for Gram-negative and Gram-positive bacteria, respectively. Left column, N-terminal Edin; right column, C-terminal Edin.

Mentions: The kinetics of edin expression closely resembles those of known AMP genes, which led us to examine whether Edin has antimicrobial properties in vitro or in vivo. To study this, we first analyzed whether Edin was able to limit bacterial growth in vitro. We overexpressed edin in S2 cells, collected the cell culture medium and incubated the medium either with E. coli or S. aureus. Medium from S2 cells transfected with an empty vector was used as a control. As shown in Figure 6A and 6B, E. coli and S. aureus grew equally well in control medium and in medium containing Edin.


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 has no broad antimicrobial properties against Gram positive or Gram negative bacteria in vitro.(A–B) Edin does no limit the growth of E. coli or S. aureus in S2 cell culture medium. S2 cells were transfected with a copper-inducible pMT-edin-V5 or an empty pMT vector, and the abilities of E. coli and S. aureus to proliferate in these mediums were analyzed. (C–G) Synthetic forms of Edin do not limit the growth of E. coli (C), E. cloacae (D), L. monocytogenes (E), E. faecalis (F) or S. aureus (G). Both N-terminal and C-terminal forms of Edin were tested. Bacteria were cultured to an OD600 nm of 0.33, incubated with synthetic Edin and the ability of the bacteria to grow was analyzed. Cecropin A and Lysozyme were used as positive controls for Gram-negative and Gram-positive bacteria, respectively. Left column, N-terminal Edin; right column, C-terminal Edin.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037153-g006: Edin has no broad antimicrobial properties against Gram positive or Gram negative bacteria in vitro.(A–B) Edin does no limit the growth of E. coli or S. aureus in S2 cell culture medium. S2 cells were transfected with a copper-inducible pMT-edin-V5 or an empty pMT vector, and the abilities of E. coli and S. aureus to proliferate in these mediums were analyzed. (C–G) Synthetic forms of Edin do not limit the growth of E. coli (C), E. cloacae (D), L. monocytogenes (E), E. faecalis (F) or S. aureus (G). Both N-terminal and C-terminal forms of Edin were tested. Bacteria were cultured to an OD600 nm of 0.33, incubated with synthetic Edin and the ability of the bacteria to grow was analyzed. Cecropin A and Lysozyme were used as positive controls for Gram-negative and Gram-positive bacteria, respectively. Left column, N-terminal Edin; right column, C-terminal Edin.
Mentions: The kinetics of edin expression closely resembles those of known AMP genes, which led us to examine whether Edin has antimicrobial properties in vitro or in vivo. To study this, we first analyzed whether Edin was able to limit bacterial growth in vitro. We overexpressed edin in S2 cells, collected the cell culture medium and incubated the medium either with E. coli or S. aureus. Medium from S2 cells transfected with an empty vector was used as a control. As shown in Figure 6A and 6B, E. coli and S. aureus grew equally well in control medium and in medium containing Edin.

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