Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms

Abstract

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

Figure 1

Induction of the diptericin gene in Drosophila adults infected by various microorganisms. (A) Drosophila adults (3–5 days old) carrying the Dipt-lacZ reporter gene (14) were pricked with a sterile needle dipped into culture pellets of distinct bacterial strains (OD of the pellet = 100), fungal spores (10¹⁰ spores per ml), or hyphae from various fungi. β-Galactosidase activity was measured 6 h after challenge at 25°C. Each bar represents the mean of several independent measurements with confidence interval (P < 5%). (B) Northern blot of total RNA extracted from one and six bacteria- or fungi-challenged wild-type (Oregon^R) male adults. The blot was successively hybridized with diptericin (Dipt) and ribosomal protein rp49 (Rp49) cDNA. Rp49 was used as an internal control for quantification of RNA. Conditions were as in A. C (control), unchallenged flies; Inj, simple injury; En.c., Enterobacter cloacae (−); S.t., Salmonella typhimurium (−); S.m., Serratia marcescens (Db1140 strain); P.a., Pseudomonas aeruginosas (−); Er.c., Erwinia carotovora; Es. c., E. coli (−); A.v., Aerococcus viridans (+); M.l., M. luteus (+); S.f., Streptococcus faecalis (+); S.a., Staphylococcus aureus (+); B.s., Bacillus subtilis (+); B.t., Bacillus thuringiensis (+) B.m., Bacillus megaterium (+); Fh, hyphal bodies; Fs, fungal spores from a mixture of Fusarium oxysporum, Neurospora crassa, and Botrytis cinerea.

Figure 2

Time-course analysis of antimicrobial gene expression after infection by various microorganisms. (A) Northern blot of total RNA extracted from female wild-type (Oregon^R) adults at different time intervals after challenge (as indicated). Adult flies infected by pricking under the same conditions were kept at 29°C. The blot was successively hybridized with the following cDNA probes: diptericin (Dipt), cecropin A1 (Cec A), attacin (Att), drosocin (Drc), defensin (Def), metchnikowin (Metch), drosomycin (Drom), and rp49 (Rp49). Unchallenged females show a low level of drosomycin gene expression due to the constitutive expression in the sperm storage structures (D. Ferrandon, personal communication). This experiment was repeated several times and yielded similar results. (B) The signals on Northern blots of Fig. 3A were quantified by a Bioimager system. The values were normalized with the corresponding value of rp49. The highest level of expression in a series was normalized as 100, and the results are given in relative activity (percent). Results obtained for defensin were similar to those for diptericin (data not shown).

Figure 3

Induction of antimicrobial peptide genes in fungi-infected adults. (A) Northern blot analysis of total RNA extracted from wild-type, Tl⁻, and Tl^D mutant female adults. Flies were anesthetized and covered with spores of B. bassiana. Flies were placed at 29°C and collected after different time intervals. (B) Northern blot analysis of total RNA extracted from wild-type female adults after natural infection by various fungi. Flies were placed at 29°C and collected 5 days later. A and B were obtained separately. C, control; d, days; Tl⁻, Tl⁶³²/Tl^1-RXA female adults; Tl^D, unchallenged Tl^10b female adults; S.i. (septic injury) adults that were challenged by pricking with a needle dipped into a mixture of E. coli and M. luteus and collected 6 h later. B.b., B. bassiana; P.f., Paecylomyces fumoroseus; A.f., A. fumigatus; M.a., Metharizium anasiplae.