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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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Related in: MedlinePlus

Knock-out of the Toll-9 locus and Toll-9 expression.(A) A complete deletion of the Toll-9 locus was generated by targeted homologous recombination using 5′ (red, II) and 3′ (red, I) homology regions; the white cDNA replaced the Toll-9 locus (black box in genomic region). (B) PCR performed on the resulting KO Toll-9 line and OregonR control. The primers used are localized on the genomic region (A, green). (C) Toll-9 expression is independent of commensal and infectious bacteria. Toll-9 expression measured by quantitative RT-PCR in conventionally reared wild-type (-1- WTCR), conventionally reared wild-type 4 hours after Erwinia carotovora carotovora 15 ingestion (-2- WTCR inf. Ecc15 -4 h-), conventionally reared wild-type 18 hours after Ecc15 ingestion (-3- WTCR inf. Ecc15 -16 h) and germ-free wild-type (-4- WTGF), in whole larvae, larval gut, whole adult and adult gut. Toll-9 is enriched in the gut of both larvae and adult, and its expression level is not dependent on commensal and infectious bacteria. Additionally, Toll-9 is no longer expressed in Toll-9−/− mutant (-5-). rp49 was used as the experimental expression standard.
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pone-0017470-g001: Knock-out of the Toll-9 locus and Toll-9 expression.(A) A complete deletion of the Toll-9 locus was generated by targeted homologous recombination using 5′ (red, II) and 3′ (red, I) homology regions; the white cDNA replaced the Toll-9 locus (black box in genomic region). (B) PCR performed on the resulting KO Toll-9 line and OregonR control. The primers used are localized on the genomic region (A, green). (C) Toll-9 expression is independent of commensal and infectious bacteria. Toll-9 expression measured by quantitative RT-PCR in conventionally reared wild-type (-1- WTCR), conventionally reared wild-type 4 hours after Erwinia carotovora carotovora 15 ingestion (-2- WTCR inf. Ecc15 -4 h-), conventionally reared wild-type 18 hours after Ecc15 ingestion (-3- WTCR inf. Ecc15 -16 h) and germ-free wild-type (-4- WTGF), in whole larvae, larval gut, whole adult and adult gut. Toll-9 is enriched in the gut of both larvae and adult, and its expression level is not dependent on commensal and infectious bacteria. Additionally, Toll-9 is no longer expressed in Toll-9−/− mutant (-5-). rp49 was used as the experimental expression standard.

Mentions: Knock-out of the Toll-9 locus has been realized by ends-out gene targeting as described in [44]. The resulting deletion covers a genomic region starting from 852 bp upstream of the Toll-9 ATG, and up to 41 bp after Toll-9 stop codon. The 3′ and 5′ homology regions I (2926 bp long) and II (3116 bp long), showed in red in Figure 1, were amplified by PCR from OregonR genomic DNA. The region I has been amplified using the following primers: F-ACATGCATGCATGTGGAAGCACTCTCGATTCAGC, R-GGGGTACCCCAGAGTTCTAGTCAGTTGTGC and contains SphI and KpnI sites respectively, whereas the region II has been amplified using the following primers: F-AGGCGCGCCTTCATCGGATACCCATTGAGG; R-CCCGTACGGGCGAGGATTCCGATAGATGCC and contains AscI and BsiWI sites. These two DNA fragments were then cloned into the pW25 vector (Drosophila Genomics Ressource Center), on both sides of the miniwhite gene whs. This pW25-Toll-9-KO construct was then transformed into y,w embryos using the standard procedure for P-element [45]. Male donor flies were crossed with y,w; hsp70-FLP, hsp70-I-SceI, nocSco/CyO females and the resulting progeny were heat-shocked at 37°C for one hour twice a day, at day 2 and 3 after egg laying. Mosaic and white eyes females were then collected from the progeny and crossed to males carrying an eyeless-FLP transgene on X chromosome. In the resulting progeny red eye males were screened. Out of 22.400 males, 26 red eye males were isolated but 7 of them were sterile. Genomic deletion of the Toll-9 locus and replacement with the whs gene were investigated by PCR, using 5F (5′-GCTGTTGACGAAGAGGGAAG-3′), 5R (GAATTGAATTGACGCTCCGT-3′), 3F (5′-GTCCGGTTGTTTTCGTGCTC-3′), 3R (5′-GTACACTTCCTTGGCTGGCG-3′), 1 (5′-CGTATTAGTATGCCTGTTCC-3′) and 21 (5′-ACAACTGACTAGAACTCTCC-3′) primers as indicated in Figure 1. Among the 19 lines established, only one showed ampification using the primer couples 5F–5R and 3F–3R, but no amplification using the primer couple 1–21 (Figure 1). This line has been isogenized using y,w flies, and kept as heterozygous in the same vial. Consequently, the control y,w has white eyes, the heterozygous y,w; Toll-9−/+ has orange eyes, and the y,w; Toll-9−/Toll-9− mutant has red eyes.


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

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

Knock-out of the Toll-9 locus and Toll-9 expression.(A) A complete deletion of the Toll-9 locus was generated by targeted homologous recombination using 5′ (red, II) and 3′ (red, I) homology regions; the white cDNA replaced the Toll-9 locus (black box in genomic region). (B) PCR performed on the resulting KO Toll-9 line and OregonR control. The primers used are localized on the genomic region (A, green). (C) Toll-9 expression is independent of commensal and infectious bacteria. Toll-9 expression measured by quantitative RT-PCR in conventionally reared wild-type (-1- WTCR), conventionally reared wild-type 4 hours after Erwinia carotovora carotovora 15 ingestion (-2- WTCR inf. Ecc15 -4 h-), conventionally reared wild-type 18 hours after Ecc15 ingestion (-3- WTCR inf. Ecc15 -16 h) and germ-free wild-type (-4- WTGF), in whole larvae, larval gut, whole adult and adult gut. Toll-9 is enriched in the gut of both larvae and adult, and its expression level is not dependent on commensal and infectious bacteria. Additionally, Toll-9 is no longer expressed in Toll-9−/− mutant (-5-). rp49 was used as the experimental expression standard.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017470-g001: Knock-out of the Toll-9 locus and Toll-9 expression.(A) A complete deletion of the Toll-9 locus was generated by targeted homologous recombination using 5′ (red, II) and 3′ (red, I) homology regions; the white cDNA replaced the Toll-9 locus (black box in genomic region). (B) PCR performed on the resulting KO Toll-9 line and OregonR control. The primers used are localized on the genomic region (A, green). (C) Toll-9 expression is independent of commensal and infectious bacteria. Toll-9 expression measured by quantitative RT-PCR in conventionally reared wild-type (-1- WTCR), conventionally reared wild-type 4 hours after Erwinia carotovora carotovora 15 ingestion (-2- WTCR inf. Ecc15 -4 h-), conventionally reared wild-type 18 hours after Ecc15 ingestion (-3- WTCR inf. Ecc15 -16 h) and germ-free wild-type (-4- WTGF), in whole larvae, larval gut, whole adult and adult gut. Toll-9 is enriched in the gut of both larvae and adult, and its expression level is not dependent on commensal and infectious bacteria. Additionally, Toll-9 is no longer expressed in Toll-9−/− mutant (-5-). rp49 was used as the experimental expression standard.
Mentions: Knock-out of the Toll-9 locus has been realized by ends-out gene targeting as described in [44]. The resulting deletion covers a genomic region starting from 852 bp upstream of the Toll-9 ATG, and up to 41 bp after Toll-9 stop codon. The 3′ and 5′ homology regions I (2926 bp long) and II (3116 bp long), showed in red in Figure 1, were amplified by PCR from OregonR genomic DNA. The region I has been amplified using the following primers: F-ACATGCATGCATGTGGAAGCACTCTCGATTCAGC, R-GGGGTACCCCAGAGTTCTAGTCAGTTGTGC and contains SphI and KpnI sites respectively, whereas the region II has been amplified using the following primers: F-AGGCGCGCCTTCATCGGATACCCATTGAGG; R-CCCGTACGGGCGAGGATTCCGATAGATGCC and contains AscI and BsiWI sites. These two DNA fragments were then cloned into the pW25 vector (Drosophila Genomics Ressource Center), on both sides of the miniwhite gene whs. This pW25-Toll-9-KO construct was then transformed into y,w embryos using the standard procedure for P-element [45]. Male donor flies were crossed with y,w; hsp70-FLP, hsp70-I-SceI, nocSco/CyO females and the resulting progeny were heat-shocked at 37°C for one hour twice a day, at day 2 and 3 after egg laying. Mosaic and white eyes females were then collected from the progeny and crossed to males carrying an eyeless-FLP transgene on X chromosome. In the resulting progeny red eye males were screened. Out of 22.400 males, 26 red eye males were isolated but 7 of them were sterile. Genomic deletion of the Toll-9 locus and replacement with the whs gene were investigated by PCR, using 5F (5′-GCTGTTGACGAAGAGGGAAG-3′), 5R (GAATTGAATTGACGCTCCGT-3′), 3F (5′-GTCCGGTTGTTTTCGTGCTC-3′), 3R (5′-GTACACTTCCTTGGCTGGCG-3′), 1 (5′-CGTATTAGTATGCCTGTTCC-3′) and 21 (5′-ACAACTGACTAGAACTCTCC-3′) primers as indicated in Figure 1. Among the 19 lines established, only one showed ampification using the primer couples 5F–5R and 3F–3R, but no amplification using the primer couple 1–21 (Figure 1). This line has been isogenized using y,w flies, and kept as heterozygous in the same vial. Consequently, the control y,w has white eyes, the heterozygous y,w; Toll-9−/+ has orange eyes, and the y,w; Toll-9−/Toll-9− mutant has red eyes.

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