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Lack of an antibacterial response defect in Drosophila Toll-9 mutant.

Narbonne-Reveau K, Charroux B, Royet J - PLoS ONE (2011)

Bottom Line: These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms.To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9.Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie du Développement de Marseille-Luminy, CNRS UMR 6216/Aix-Marseille II University, Campus de Luminy, Marseille, France.

ABSTRACT
Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.

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Systemic AMP production is independent of Toll-9 function.Quantitative RT-PCR analysis of AMPs induction after Gram-negative bacteria ingestion. Diptericin (A. C, E), Drosomycin (B, D) and Drosomycin3 (F) expression upon Ecc15 oral ingestion in Toll-9−/− whole larvae (A, B), whole adults (C, D), and adult gut (E, F), and in relishE20 whole adult (C, D), and adult guts (E), compared to control. rp49 was used as the experimental expression standard. Relative AMP/rp49 ratios of unchallenged controls were set to 1 to indicate fold-induction.
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pone-0017470-g006: Systemic AMP production is independent of Toll-9 function.Quantitative RT-PCR analysis of AMPs induction after Gram-negative bacteria ingestion. Diptericin (A. C, E), Drosomycin (B, D) and Drosomycin3 (F) expression upon Ecc15 oral ingestion in Toll-9−/− whole larvae (A, B), whole adults (C, D), and adult gut (E, F), and in relishE20 whole adult (C, D), and adult guts (E), compared to control. rp49 was used as the experimental expression standard. Relative AMP/rp49 ratios of unchallenged controls were set to 1 to indicate fold-induction.

Mentions: The data above suggest that contrary to what has been proposed earlier, Toll-9 is not required to maintain a basal level of AMPs production in vivo. We then tested whether Toll-9 is necessary to regulate immune gene induction after microbial challenge. As Toll-9 is mainly expressed in the gut, we used the Ecc15 oral infection model. First, we observed that the survival of Toll-9−/− mutants was not affected by Ecc15 oral infection (Figure S1). We next checked induction levels of Diptericin and Drosomycin in whole larvae and adult, and Diptericin and Drosomycin3 expression in the adult gut (Figure 6). Since antimicrobial peptides gut induction has been shown to depend essentially on the Imd pathway, we used relishE20 as a control. Upon Ecc15 oral infection, Diptericin expression increased up to 120-fold in whole larvae (Figure 6A), 25-fold in whole adults (Figure 6C) and 6 times in adult guts (Figure 6E). Similar induction levels were obtained in Toll-9−/− mutant background (Figure 6A, C, E, for each experiment, P≥0.25), whereas, as expected, Diptericin induction was abolished in a relishE20 mutant (Figure 6C, E). Similarly, no significant variation was observed for Drosomycin and Drosomycin3 induction between wild-type and Toll-9−/− mutants (Figure 6B, D, F). These results were confirmed by using reporter lines for Cecropin, Drosomycin and Diptericin (Cec-GFP, Drs-GFP and Dpt-cherry, respectively), both in the larvae (Figure 5C–F) and in the adult (data not shown). To complete this analysis, we performed oral infection using other bacteria species such as Pseudomonas entomophila, Serratia marcescens and Lactobacillus plantarum. Twenty hours following oral ingestion of these bacteria, Diptericin mRNAs induction was not significantly different in Toll9−/− versus control midguts (Figure S2, for each experiment, P≥0.1). From these experiments we concluded that the basal and oral infection-induced antimicrobial gene levels are unaffected by Toll-9−/− inactivation. This suggests that Toll-9 does not participate in maintaining basal expression levels of antimicrobial peptides genes in normal conditions or after oral immune challenge.


Lack of an antibacterial response defect in Drosophila Toll-9 mutant.

Narbonne-Reveau K, Charroux B, Royet J - PLoS ONE (2011)

Systemic AMP production is independent of Toll-9 function.Quantitative RT-PCR analysis of AMPs induction after Gram-negative bacteria ingestion. Diptericin (A. C, E), Drosomycin (B, D) and Drosomycin3 (F) expression upon Ecc15 oral ingestion in Toll-9−/− whole larvae (A, B), whole adults (C, D), and adult gut (E, F), and in relishE20 whole adult (C, D), and adult guts (E), compared to control. rp49 was used as the experimental expression standard. Relative AMP/rp49 ratios of unchallenged controls were set to 1 to indicate fold-induction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046252&req=5

pone-0017470-g006: Systemic AMP production is independent of Toll-9 function.Quantitative RT-PCR analysis of AMPs induction after Gram-negative bacteria ingestion. Diptericin (A. C, E), Drosomycin (B, D) and Drosomycin3 (F) expression upon Ecc15 oral ingestion in Toll-9−/− whole larvae (A, B), whole adults (C, D), and adult gut (E, F), and in relishE20 whole adult (C, D), and adult guts (E), compared to control. rp49 was used as the experimental expression standard. Relative AMP/rp49 ratios of unchallenged controls were set to 1 to indicate fold-induction.
Mentions: The data above suggest that contrary to what has been proposed earlier, Toll-9 is not required to maintain a basal level of AMPs production in vivo. We then tested whether Toll-9 is necessary to regulate immune gene induction after microbial challenge. As Toll-9 is mainly expressed in the gut, we used the Ecc15 oral infection model. First, we observed that the survival of Toll-9−/− mutants was not affected by Ecc15 oral infection (Figure S1). We next checked induction levels of Diptericin and Drosomycin in whole larvae and adult, and Diptericin and Drosomycin3 expression in the adult gut (Figure 6). Since antimicrobial peptides gut induction has been shown to depend essentially on the Imd pathway, we used relishE20 as a control. Upon Ecc15 oral infection, Diptericin expression increased up to 120-fold in whole larvae (Figure 6A), 25-fold in whole adults (Figure 6C) and 6 times in adult guts (Figure 6E). Similar induction levels were obtained in Toll-9−/− mutant background (Figure 6A, C, E, for each experiment, P≥0.25), whereas, as expected, Diptericin induction was abolished in a relishE20 mutant (Figure 6C, E). Similarly, no significant variation was observed for Drosomycin and Drosomycin3 induction between wild-type and Toll-9−/− mutants (Figure 6B, D, F). These results were confirmed by using reporter lines for Cecropin, Drosomycin and Diptericin (Cec-GFP, Drs-GFP and Dpt-cherry, respectively), both in the larvae (Figure 5C–F) and in the adult (data not shown). To complete this analysis, we performed oral infection using other bacteria species such as Pseudomonas entomophila, Serratia marcescens and Lactobacillus plantarum. Twenty hours following oral ingestion of these bacteria, Diptericin mRNAs induction was not significantly different in Toll9−/− versus control midguts (Figure S2, for each experiment, P≥0.1). From these experiments we concluded that the basal and oral infection-induced antimicrobial gene levels are unaffected by Toll-9−/− inactivation. This suggests that Toll-9 does not participate in maintaining basal expression levels of antimicrobial peptides genes in normal conditions or after oral immune challenge.

Bottom Line: These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms.To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9.Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biologie du Développement de Marseille-Luminy, CNRS UMR 6216/Aix-Marseille II University, Campus de Luminy, Marseille, France.

ABSTRACT
Toll and Toll-like receptors represent families of receptors involved in mediating innate immunity response in insects and mammals. Although Drosophila proteome contains multiple Toll paralogs, Toll-1 is, so far, the only receptor to which an immune role has been attributed. In contrast, every single mammalian TLR is a key membrane receptor upstream of the vertebrate immune signaling cascades. The prevailing view is that TLR-mediated immunity is ancient. Structural analysis reveals that Drosophila Toll-9 is the most closely related to vertebrate TLRs and utilizes similar signaling components as Toll-1. This suggests that Toll-9 could be an ancestor of TLR-like receptors and could have immune function. Consistently, it has been reported that over-expression of Toll-9 in immune tissues is sufficient to induce the expression of some antimicrobial peptides in flies. These results have led to the idea that Toll-9 could be a constitutively active receptor that maintain significant levels of antimicrobial molecules and therefore provide constant basal protection against micro-organisms. To test theses hypotheses, we generated and analyzed phenotypes associated with a complete loss-of-function allele of Toll-9. Our results suggest that Toll-9 is neither required to maintain a basal anti-microbial response nor to mount an efficient immune response to bacterial infection.

Show MeSH
Related in: MedlinePlus