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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 microbial binding.500 µl of Edin-V5 containing medium were incubated with 1 ml of a bacterial suspension of live E. coli (E.c.), Serratia marcescens (S.m), Staphylococcus epidermidis (S.e.), Enterococcus faecalis (E.f.), Listeria monocytogenes (L.m.), Micrococcus luteus (M.l.), Saccharomyces cerevisiae (S.c.) or S. aureus (S.a) for 1 h with mild agitation at +4°C. Latex beads treated with BSA were used as a control. The samples were then centrifuged and the pellet was washed. Edin bound to microbes was detached by adding 20 µl of SDS-PAGE loading buffer, boiled for 10 minutes, electrophoresed on SDS-PAGE and detected using a V5 antibody. The first lane of each blot is a control sample containing 20 µl of Edin-V5 medium. The following lanes contain 30 µl of the medium incubated with the indicated microbe.
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pone-0037153-g003: The effect of Edin on microbial binding.500 µl of Edin-V5 containing medium were incubated with 1 ml of a bacterial suspension of live E. coli (E.c.), Serratia marcescens (S.m), Staphylococcus epidermidis (S.e.), Enterococcus faecalis (E.f.), Listeria monocytogenes (L.m.), Micrococcus luteus (M.l.), Saccharomyces cerevisiae (S.c.) or S. aureus (S.a) for 1 h with mild agitation at +4°C. Latex beads treated with BSA were used as a control. The samples were then centrifuged and the pellet was washed. Edin bound to microbes was detached by adding 20 µl of SDS-PAGE loading buffer, boiled for 10 minutes, electrophoresed on SDS-PAGE and detected using a V5 antibody. The first lane of each blot is a control sample containing 20 µl of Edin-V5 medium. The following lanes contain 30 µl of the medium incubated with the indicated microbe.

Mentions: To investigate in a more direct way if Edin binds microbes, we incubated Edin-containing cell culture medium with live E. coli, Serratia marcescens, Staphylococcus epidermidis, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus, Saccharomyces cerevisiae and S. aureus. Latex beads (carboxylated polystyrene), which are expected to bind all kinds of proteins to some extent, were used as a positive control. The microbial suspensions were incubated with 500 µl of Edin-containing medium at +4°C after which the microbes were pelleted and washed with PBS. Finally, the pellets were suspended and boiled in an SDS-PAGE sample buffer to detach bound Edin from the microbes before electrophoresis. Next, the proteins were transferred onto nitrocellulose membranes and Edin was detected using an anti-V5 antibody. As a reference, 20 µl of Edin-containing medium was loaded into the first lane. Therefore, if Edin attached efficiently to the indicated microbe, much more Edin should be detected in the samples (500 µl Edin-containing medium used) compared to the reference lane (20 µl Edin-containing medium). As shown in Figure 3 (the rightmost lanes), carboxylated latex beads, i.e. the positive control, bound Edin. In contrast, virtually no Edin was bound to the tested Gram-negative bacteria, E. coli and S. marcescens. Furthermore, only a faint signal was detected with the Gram-positive bacteria S. epidermidis, E. faecalis, L. monocytogenes, M. luteus and S. aureus, and with the baker's yeast S. cerevisiae as compared to the reference lane (ctrl in Figure 3). Based on these results, we conclude that Edin does not strongly bind any of the tested microbes.


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 microbial binding.500 µl of Edin-V5 containing medium were incubated with 1 ml of a bacterial suspension of live E. coli (E.c.), Serratia marcescens (S.m), Staphylococcus epidermidis (S.e.), Enterococcus faecalis (E.f.), Listeria monocytogenes (L.m.), Micrococcus luteus (M.l.), Saccharomyces cerevisiae (S.c.) or S. aureus (S.a) for 1 h with mild agitation at +4°C. Latex beads treated with BSA were used as a control. The samples were then centrifuged and the pellet was washed. Edin bound to microbes was detached by adding 20 µl of SDS-PAGE loading buffer, boiled for 10 minutes, electrophoresed on SDS-PAGE and detected using a V5 antibody. The first lane of each blot is a control sample containing 20 µl of Edin-V5 medium. The following lanes contain 30 µl of the medium incubated with the indicated microbe.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351453&req=5

pone-0037153-g003: The effect of Edin on microbial binding.500 µl of Edin-V5 containing medium were incubated with 1 ml of a bacterial suspension of live E. coli (E.c.), Serratia marcescens (S.m), Staphylococcus epidermidis (S.e.), Enterococcus faecalis (E.f.), Listeria monocytogenes (L.m.), Micrococcus luteus (M.l.), Saccharomyces cerevisiae (S.c.) or S. aureus (S.a) for 1 h with mild agitation at +4°C. Latex beads treated with BSA were used as a control. The samples were then centrifuged and the pellet was washed. Edin bound to microbes was detached by adding 20 µl of SDS-PAGE loading buffer, boiled for 10 minutes, electrophoresed on SDS-PAGE and detected using a V5 antibody. The first lane of each blot is a control sample containing 20 µl of Edin-V5 medium. The following lanes contain 30 µl of the medium incubated with the indicated microbe.
Mentions: To investigate in a more direct way if Edin binds microbes, we incubated Edin-containing cell culture medium with live E. coli, Serratia marcescens, Staphylococcus epidermidis, Enterococcus faecalis, Listeria monocytogenes, Micrococcus luteus, Saccharomyces cerevisiae and S. aureus. Latex beads (carboxylated polystyrene), which are expected to bind all kinds of proteins to some extent, were used as a positive control. The microbial suspensions were incubated with 500 µl of Edin-containing medium at +4°C after which the microbes were pelleted and washed with PBS. Finally, the pellets were suspended and boiled in an SDS-PAGE sample buffer to detach bound Edin from the microbes before electrophoresis. Next, the proteins were transferred onto nitrocellulose membranes and Edin was detected using an anti-V5 antibody. As a reference, 20 µl of Edin-containing medium was loaded into the first lane. Therefore, if Edin attached efficiently to the indicated microbe, much more Edin should be detected in the samples (500 µl Edin-containing medium used) compared to the reference lane (20 µl Edin-containing medium). As shown in Figure 3 (the rightmost lanes), carboxylated latex beads, i.e. the positive control, bound Edin. In contrast, virtually no Edin was bound to the tested Gram-negative bacteria, E. coli and S. marcescens. Furthermore, only a faint signal was detected with the Gram-positive bacteria S. epidermidis, E. faecalis, L. monocytogenes, M. luteus and S. aureus, and with the baker's yeast S. cerevisiae as compared to the reference lane (ctrl in Figure 3). Based on these results, we conclude that Edin does not strongly bind any of the tested microbes.

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