Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity

Abstract

In innate immunity, pattern recognition molecules recognize cell wall components of microorganisms and activate subsequent immune responses, such as the induction of antimicrobial peptides and melanization in Drosophila. The diaminopimelic acid (DAP)-type peptidoglycan potently activates imd-dependent induction of antibacterial peptides. Peptidoglycan recognition protein (PGRP) family members act as pattern recognition molecules. PGRP-LC loss-of-function mutations affect the imd-dependent induction of antibacterial peptides and resistance to Gram-negative bacteria, whereas PGRP-LE binds to the DAP-type peptidoglycan, and a gain-of-function mutation induces constitutive activation of both the imd pathway and melanization. Here, we generated PGRP-LE null mutants and report that PGRP-LE functions synergistically with PGRP-LC in producing resistance to Escherichia coli and Bacillus megaterium infections, which have the DAP-type peptidoglycan. Consistent with this, PGRP-LE acts both upstream and in parallel with PGRP-LC in the imd pathway, and is required for infection-dependent activation of melanization in Drosophila. A role for PGRP-LE in the epithelial induction of antimicrobial peptides is also suggested.

Figure 1

Survival rate of PGRP-LE mutant flies after different types of infections. (A–F) Survival rate of wild-type flies (wt), PGRP-LE¹¹², PGRP-LC⁷⁴⁵⁴, imd¹, and the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant (PGRP-LE¹¹²;+; PGRP-LC⁷⁴⁵⁴) infected with the indicated bacteria. In (F), the survival rate of Relish^E20 is also presented. (G) Survival rate of wild type, Relish^E20, the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant, and the double PGRP-LE¹¹²/PGRP-LC^ΔE mutant against E. coli infection. (H) Survival rate of wild type, Relish^E20, the double PGRP-LE¹¹²/Relish^E20 mutant, and the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant against E. coli infection. (I) Survival rate of wild type, J4, the double J4/PGRP-LC⁷⁴⁵⁴ mutant, and the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant against E. coli infection. The survival experiments were performed at 25°C.

Figure 2

Epistatic analyses of PGRP-LE with PGRP-LC on antimicrobial peptide gene expression. Under the control of c564-GAL4, GFP, PGRP-SA, PGRP-LCx, and PGRP-LE were overexpressed in the wild-type (+), PGRP-LE¹¹², PGRP-LC^ΔE, and Relish^E20 larvae. The amount of mRNA of Diptericin (A), Attacin (B), and Drosomycin (C), and rp49 internal control was quantified by real-time RT–PCR in each sample.

Figure 3

Expression of seven classes of antimicrobial peptide genes in different mutants after various bacterial challenges. After B. subtilis (A), E. faecalis (B), and E. coli (C) infection, the amount of mRNA of seven classes of inducible antimicrobial peptides, Diptericin (Dip), Drosomycin (Drs), Attacin (Att), Cecropin A (CecA), Metchnikowin (Mtk), Drosocin (Dro), and Defensin (Def), and the rp49 internal control in the indicated mutant flies, wild type (wt), PGRP-LE¹¹², PGRP-LC⁷⁴⁵⁴, spätzle^rm7, imd¹, and the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant, was quantified by real-time RT–PCR. As a negative control, pyrogen-free saline was used (D). Each experiment is representative of at least two independent experiments.

Figure 4

Activation of Drosomycin promoter in various mutants after natural infection of E. carotovora carotovora. The number of Drs-GFP-expressing larvae was counted in wild type (wt), PGRP-LE¹¹², PGRP-LC⁷⁴⁵⁴, and the double PGRP-LE¹¹²/PGRP-LC⁷⁴⁵⁴ mutant. Bars indicate standard deviation of duplicate measurements.

Figure 5

Requirement of PGRP-LE on the infection-dependent activation of the proPO cascade. (A, B) PO activity in the hemolymph after E. coli infection. The hemolymph was collected from wild-type (wt), PGRP-LE¹¹², UAS-PGRP-LE/+; hs-GAL4/+, and PGRP-LE¹¹²; UAS-PGRP-LE/+; hs-GAL4/+ flies. (C–F) Melanization at the injury site (black arrowhead) of the indicated flies after E. coli challenge. White arrowhead indicates the cuticle defect (D). (E, F) Higher magnifications of (C, D) respectively. (G) PO activity in various mutant larvae. PO activity was assayed with homogenates of wild-type (wt), GS1068; +; hs-GAL4/+, GS1068; UAS-Serpin27A/+; hs-GAL4/+, GS1068; serpin27A¹; hs-GAL4/+, GS1068; Bc¹; hs-GAL4/+, GS1068; imd¹; hs-GAL4/+, serpin27A¹, serpin27A¹Bc¹, PGRP-LE¹¹²; serpin27A¹ larvae. (H–J) The PGRP-LE-mediated induction of antimicrobial peptide genes. The amount of mRNA of Diptericin (H), Attacin (I), and Drosomycin (J), and rp49 internal control was quantified by real-time RT–PCR in GS1068; +; hs-GAL4/+, GS1068; +; hs-GAL4/UAS-Serpin27A, GS1068; c564-GAL4/+, GS1068; c564-GAL4/+; UAS-Serpin27A/+ larvae. mRNA was recovered from the larvae at 14 h after heat shock (31°C, 60 min). (K, L) The PGRP-LE-mediated melanization of GS1068; c564-GAL4/+ larvae (K) and GS1068; c564-GAL4/+; spätzle^rm7 larvae (L). Arrowheads indicate melanization. (M) PGRP-LE-mediated activation of the proPO cascade. PO activity was assayed with homogenates of wild-type larvae (wt), spätzle^rm7, GS1068; c564-GAL4/+ larvae, and GS1068; c564-GAL4/+; spätzle^rm7 larvae. Bars indicate standard deviation of duplicate measurements.

Figure 6

Non-cell autonomous effects of PGRP-LE on the activation of antimicrobial peptide genes in systemic and epithelial reactions. (A, B) Western blotting analyses using antibody against PGRP-LE (A) and Hrp48 (B). The homogenates (20 μg) prepared from wild-type (wt), PGRP-LE¹¹², and UAS-PGRP-LE/+; hs-GAL4/+ larvae after heat shock (35°C, 20 min) were applied to the analysis. The hemolymph (plasma) fraction (35 μg, S) and the hemocyte fraction (P) prepared from wild-type (wt) and PGRP-LE¹¹² larvae were analyzed. The arrowheads indicate 45-, 30-, and 21.5-kDa proteins (A). Molecular size markers are indicated on the right. (C, D) The Drs-GFP expression in GS1068; hs-GAL4/Drs-GFP larvae (C) and GS1068; imd¹; hs-GAL4/Drs-GFP larvae (D) 12 h after heat shock (35°C, 20 min). (E) Non-cell autonomous effects of PGRP-LE on the expression of Dpt-lacZ in the fat body. (F) Cell autonomous effects of Imd on the expression of Dpt-lacZ in the fat body. (G) Non-cell autonomous effects of PGRP-LE on the expression of Drs-lacZ in the trachea. The overexpression of PGRP-LE and Imd was monitored by the expression of GFP (green). The expression of antimicrobial peptide genes was monitored by the expression of reporter genes using Cy3-labeled antibody (red). The GFP, Cy3, and DAPI (nuclear staining, blue) signals are merged.

Figure 7

The epithelial induction of PGRP-LE is sufficient to activate the epithelial response. (A, B) Expression of Dpt-lacZ in the fat body (A) and Drs-GFP (B) in Dpt-lacZ, Drs-GFP; c564-GAL4/UAS-PGRP-LE larvae. (C, D) Expression of Dpt-lacZ in the fat body (C) and Drs-GFP (D) in Dpt-lacZ, Drs-GFP; NP2610-GAL4/UAS-PGRP-LE larvae.

Figure 8

Localization of PGRP-LE in the trachea. (A–F) Immunostaining was performed in wild type (wt, A, B, E) and PGRP-LE¹¹² (C, D, F) using anti-PGRP-LE antibody. The antibody staining is merged with DAPI staining (A, C). The bright-field micrograph is merged with DAPI staining (B, D). Confocal analysis in wild type (E) and PGRP-LE¹¹² (F) using anti-PGRP-LE antibody. Arrowheads indicate PGRP-LE signal. (G) Western blotting analysis was performed with trachea and fat body homogenates prepared from wild type (wt) and PGRP-LE¹¹² using anti-PGRP-LE antibody. Arrowhead indicates 45-kDa protein.