Overexpression of a pattern-recognition receptor, peptidoglycan-recognition protein-LE, activates imd/relish-mediated antibacterial defense and the prophenoloxidase cascade in Drosophila larvae

Abstract

In Drosophila, microbial infection activates an antimicrobial defense system involving the activation of proteolytic cascades in the hemolymph and intracellular signaling pathways, the immune deficiency (imd) and Toll pathways, in immune-responsive tissues. The mechanisms for microbial recognition are largely unknown. We report that, in larvae, the imd-mediated antibacterial defense is activated by peptidoglycan-recognition protein (PGRP)-LE, a PGRP-family member in Drosophila. Consistent with this, PGRP-LE binds to the diaminopimelic acid-type peptidoglycan, a cell-wall component of the bacteria capable of activating the imd pathway, but not to the lysine-type peptidoglycan. Moreover, PGRP-LE activates the prophenoloxidase cascade, a proteolytic cascade in the hemolymph. Therefore, PGRP-LE acts as a pattern-recognition receptor to the diaminopimelic acid-type peptidoglycan and activates both the proteolytic cascade and intracellular signaling in Drosophila immunity.

Figure 1

Induction of Diptericin by forced expression of PGRP-LE in Drosophila. (A and B) Histochemical staining of fat-body β-galactosidase activity in third-instar larvae carrying the Diptericin-lacZ reporter gene. Significant expression of the lacZ gene is observed in the fat body of transheterozygous larvae of hs-GAL4;GS1068 (A), but not in the fat body of control larvae with hs-GAL4 and GS insertions (B), in the absence of microbial infection. The transheterozygotes and control larvae were incubated at 29°C for 2 h and stained with 5-bromo-4-chloro-3-indolyl β-d-galactoside after recovery for 12 h at 25°C. (C and D) The forced expression of PGRP-LE and induction of Diptericin in GS1068 via GAL4/UAS system. The amount of mRNA of PGRP-LE (C), Diptericin (D), and rp49 internal control was quantified by real-time RT-PCR with a LightCycler (Roche Diagnostics). The RNA copy numbers were standardized against that of the RNA coding rp49 in each sample and are shown. The WT larvae (lane 1), GS1068 larvae (lane 2), and hs-GAL4;GS1068 transheterozygous larvae (lane 3) were incubated at 35°C for 20 min and allowed to recover at 25°C for 12 h. (E and F) Induction of endogenous Diptericin gene by the forced expression of PGRP-LE. The FLAG-tagged PGRP-LE was induced by the GAL4/UAS system. The UAS-PGRP-LE larvae and hs-GAL4;UAS-PGRP-LE transheterozygous larvae were incubated at 35°C for 20 min and allowed to recover at 25°C for 12 h. The protein (20 μg of each) of the homogenate of UAS-PGRP-LE larvae (lane 1) and the homogenate of hs-GAL4;UAS-PGRP-LE larvae (lane 2) were probed with anti-FLAG-tag antibody (E). Molecular size markers are indicated on the left. The amount of mRNA of Diptericin and rp49 internal control was quantified by real-time RT-PCR (F). Lane 1, UAS-PGRP-LE larvae; lane 2, hs-GAL4;UAS-PGRP-LE transheterozygous larvae. The results of real-time PCR analyses were confirmed by three independent experiments.

Figure 2

Activation of imd-mediated antibacterial response by forced expression of PGRP-LE in Drosophila. (A–C) The quantitative analyses of mRNA of antimicrobial peptide genes with various mutant backgrounds. The transheterozygous larvae were incubated at 35°C for 20 min and allowed to recover at 25°C for 12 h. The amount of mRNA of Diptericin (A), Drosomycin (B), Attacin (C), and rp49 internal control was quantified by real-time RT-PCR in each sample. The copy numbers of antibacterial gene mRNA were standardized against that of rp49 in each sample. (−) Unchallenged wild-type larvae. (+) Immune challenge was performed by pricking wild-type larvae with a fine needle dipped into a concentrated culture of Escherichia coli, and RNA was prepared 6 h after infection. (PGRP-LE) GS1068/+;hs-GAL4/+. (PGRP-LE, imd⁻) GS1068/+;imd/imd;hs-GAL4/+. (PGRP-LE, Relish⁻) GS1068/+;hs-GAL4/+;Relish ^E38/Relish ^E38. (PGRP-LE, J4) GS1068/+; J4/J4; hs-GAL4/+. The results were confirmed by three independent experiments. (D and E) Differential induction of antimicrobial peptide genes by PGRP-LE in larvae and adults. The amount of mRNA of antimicrobial peptide (AMP) genes, Diptericin (Dpt), Drosomycin (Drs), and Attacin (Att), and rp49 in c564;GS1068 transheterozygous larvae was quantified by real-time RT-PCR (D). Some adults of a weak UAS-PGRP-LE line escaped from pupal lethality in combination with hs-GAL4. The transheterozygous adults were incubated at 35°C for 20 min and allowed to recover at 25°C for 3 h. The amount of mRNA of Diptericin (Dpt), Drosomycin (Drs), Attacin (Att), and rp49 was quantified by real-time RT-PCR (E). (−) hs-GAL4;UAS-lacZ. (+) hs-GAL4;UAS-PGRP-LE transheterozygous adults. (F–H) PGRP-LE-mediated induction of Drosomycin-GFP in the epithelial tissue. The UAS-PGRP-LE;Drs-GFP (F) and hs-GAL4;UAS-PGRP-LE;Drs-GFP (G) transheterozygous larvae were incubated at 35°C for 20 min and allowed to recover at 25°C for 12 h. The expression of Drs-GFP is induced in the trachea (G) and salivary glands (out of focus in G) by the forced expression of PGRP-LE. Immune challenge induces Drs-GFP expression in the fat body of Drs-GFP larvae (H).

Figure 3

Specific binding of PGRP-LE to the DAP-type peptidoglycan. The DAP-type peptidoglycan of L. plantarum ATCC 8014, the Lys-type peptidoglycans of S. epidermidis ATCC 155, the Lys-type peptidoglycan of S. aureus (Wako), and the DAP-type peptidoglycan-containing cell wall of N. calcarea ATCC 17876 were used for binding assay. Approximately 0.5 μg of purified recombinant PGRP-LE (lane 1) was incubated with 0.32 mg of insoluble peptidoglycans of S. epidermidis (lanes 2 and 3), S. aureus (lanes 4 and 5), and L. plantarum (lanes 6 and 7) and the same amount of the DAP-type peptidoglycan-containing cell wall of N. calcarea ATCC 17876 (lanes 8 and 9). Unbound protein (lanes 2, 4, 6, and 8) isolated from the soluble fraction and bound protein (lanes 3, 5, 7, and 9) recovered after washing the peptidoglycans and the cell wall with Tris-maleate buffer containing 1 mol/liter NaCl and 1 mol/liter NaCl plus 0.2% Tween 20 were analyzed by Western blot analysis by using anti-penta-histidine antibody (Qiagen). Molecular size markers are indicated on the left.

Figure 4

Induction of melanization by forced expression of PGRP-LE in Drosophila. Localized melanization (arrow) is induced in immune-challenged, WT larvae (A) but not in unchallenged larvae (B). When ubiquitous PGRP-LE expression was induced by heat shock-inducible GAL4, localized melanization (arrow) was observed in the cuticle of the larvae (C). After the appearance of the localized melanization, the larvae died of melanization in the whole body. The transheterozygous larvae of hs-GAL4;GS1068 were incubated at 37°C for 1 h and allowed to recover at 25°C. Pigmented particles were observed in the hemolymph of transheterozygous larvae of c564;GS1068 (D) but not in the hemolymph of control larvae of c564;UAS-lacZ (E).