The Drosophila caspase Dredd is required to resist gram-negative bacterial infection

Abstract

The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. We have isolated loss-of-function mutations in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by gram-negative bacteria. Our results demonstrate that Dredd regulates antibacterial peptide gene expression, and we propose that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist gram-negative bacterial infections.

Fig. 1. dredd is required for diptericin expression in larvae and adults. (A) Bacterial infection induces the expression of the diptericin–GFP reporter gene in the fat bodies of wild-type (WT) larvae. This induction is blocked by the B118 mutation. (B) Northern blot analysis of total RNA extracted from adult flies infected with a mixture of Gram-positive (Micrococcus luteus) and Gram-negative (Escherichia coli) bacteria shows that all five alleles of dredd (B118, F64, D44, D55, L23) and the Df(1)dredd^D3 (D3) deficiency completely block diptericin expression. diptericin is weakly expressed in the EP-1412 line that carries a P-element insertion in the dredd gene. diptericin expression is restored in B118 and D3 flies carrying the P[dredd⁺] transgene. rp49 expression was monitored as a loading control. NI: non-infected. (C) A genomic map of the dredd locus (Chen et al., 1998) showing the P-element insertion site (EP-1412), the sequences deleted in deficiencies Df(1)R194 and Df(1)dredd^D3, and the genomic DNA contained in the P[dredd⁺] transgene. (D) Histochemical staining for lacZ activity shows that a P[dredd–lacZ] reporter gene is constitutively expressed in the fat bodies of uninfected larvae (left) and adults (right). (E) The five EMS-induced alleles of dredd each contain a single point mutation that generates the indicated changes in the Dredd protein.

Fig. 2. Dredd regulates the expression of antibacterial genes. (A) A time course of antimicrobial gene expression in different mutant adults infected with a mixture of E. coli and M. luteus shows that dredd, relish and imd predominantly control the expression of the antibacterial genes (diptericin, cecropin A, attacin, defensin). The northern blot was performed with total RNA extracted from wild type, and dredd, imd, rel and spz mutant adults at different time intervals after challenge (as indicated in hours). Flies were incubated at 25°C. The blot was successively hybridized with the following cDNA probes: diptericin (dipt), cecropin A (cecA), attacin (att), defensin (def), metchnikowin (metk), drosomycin (drom) and rp49. (B) The quantification of metchnikowin and drosomycin expression in different mutant adults collected 6 h after infection by either Gram-negative (–, E. coli) or Gram-positive (+, M. luteus) bacteria indicates that dredd and relish are required for the expression of these genes after Gram-negative, but not Gram-positive bacterial infection. As observed for the other antimicrobial genes the effect of the imd mutation on the expression of these genes is weaker than the dredd and relish mutations (Figure 2A). The signals from a northern blot were quantified with a Bio-Imager system and the levels of metchnikowin and drosomycin expression were normalized with the corresponding value of the rp49 signal. (C) The high levels of drosomycin gene expression in uninfected Tl^10b and dredd^B118;Tl^10b adults demonstrate that dredd is not required for the Tl^10b-driven constitutive expression of drosomycin.

Fig. 3. dredd is required for resistance to Gram-negative bacterial infection. (A) Mutations in dredd, relish and imd render adult flies highly susceptible to E. coli infection. (B) Only flies carrying both the imd and spz mutation are sensitive to M. luteus infection. (C) The spz gene is required for resistance to natural infection by Beauveria bassiana. The survival rate of wild-type (Canton^S, diamonds), imd (×), spz^rm7 (circles), dredd^B118 (squares), rel^E20 (triangles) and imd;spz^rm7 (asterisks) infected flies are presented with confidence intervals (p <5%). One hundred to 200 adults, aged 2–4 days, were pricked and transferred at 29°C to a fresh vial every 3 days. Adults were pricked with a needle previously dipped into either E. coli (A), M. luteus (B) or naturally infected by B. bassiana (C). The mutated flies tested here exhibited >80% survival 100 h after challenge by a clean injury (data not shown).