Diversity of innate immune recognition mechanism for bacterial polymeric meso-diaminopimelic acid-type peptidoglycan in insects

Abstract

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects.

FIGURE 1.

Polymeric DAP-type PG forms a complex with PGRP-SA and induces activation of SPE zymogen. A, the ability of Tenebrio PGRP-SA to bind to polymeric Lys-type and DAP-type PGs. Lanes 1 and 2, Tenebrio PGRP-SA with S. aureus and M. luteus Lys-type PGs, respectively. Lanes 3 and 4, PGRP-SA with E. coli and B. subtilis polymeric DAP-type PGs, respectively. Tenebrio PGRP-SA supernatant and precipitate were analyzed by SDS-PAGE. B, elution patterns of gel filtration column after loading Tenebrio PGRP-SA only (panel a), PGRP-SA with polymeric Lys-type PG (panel b), PGRP-SA with polymeric DAP-type PG (panel c), and PGRP-SA with lysozyme-treated monomeric DAP-type PG (panel d). The boxes indicate the SDS/PAGE analyses patterns of the fractions after column. C, measurement of amidase activity of activated SPE. Lane 2, a mixture of Lys-type PG with PGRP-SA and downstream factors (GNBP1, MSP, SAE, and SPE zymogens). Lanes 3–6 indicate the amidase activities of a mixture of fractions A–D in Fig. 6B with downstream factors in the presence of Ca²⁺. Tm, T. molitor.

FIGURE 2.

In vitro reconstitution experiments for the activation of pro-MSP and processing of pro-Spätzle by polymeric DAP-type PG. A, mixture of purified Tenebrio PGRP-SA, GNBP1, and MSP zymogen in the presence of S. aureus Lys-type PG (lanes 2 and 3), B. subtilis polymeric DAP-type PG (lanes 4 and 5), and E. coli DAP-type PG (lanes 6 and 7) were incubated for 60 min and then analyzed by Western blotting with an anti-MSP antibody. The 82-kDa pro-MSP and the 35-kDa activated MSP are indicated with arrows. In the absence of PGRP-SA, pro-MSP was not cleaved (lanes 2, 4, and 6). B, mixture of Tenebrio PGRP-SA, GNBP1, MSP, SAE, SPE zymogens, and pro-Spätzle (SPZ) in the presence of S. aureus Lys-type PG (lanes 2 and 3), B. subtilis polymeric DAP-type PG (lanes 4 and 5), and E. coli DAP-type PG (lanes 6 and 7) were incubated for 60 min and then analyzed by Western blotting with an affinity-purified anti-SPZ antibody. The 30-kDa pro-SPZ and the 12-kDa processed SPZ are indicated with arrows. As a control, when eight components, such as Lys-type PG·PGRP-SA·GNBP1·MSP·SAE·SPE·Spätzle, were incubated together, the cleaved 12-kDa SPZ was generated (lane 3). In the absence of PGRP-SA, pro-SPZ was not converted to the processed SPZ (lanes 2, 4, and 6). Tm, T. molitor; Ab, antibody; S. a, S. aureus; B. s, B. subtilis; E. c, E. coli.

FIGURE 3.

Polymeric DAP-type PG induces melanin and AMP synthesis. One hundred nanograms of Lys-type PG (A), polymeric DAP-type PG (B), or lysozyme-treated DAP-type PG (C) was injected into ten Tenebrio larvae, respectively. Four μl of insect saline was injected as a control (D). Within 18 h, the appearance of melanin pigment was examined. Antibacterial activities after injection of PGs (50 ng) are shown against S. aureus (E) and E. coli (F), respectively. Columns 1 and 5, columns 2 and 6, columns 3 and 7, and columns 4 and 8 are injected with insect saline (4 μl), Lys-type PG (50 ng), polymeric DAP-type PG (50 ng), and lysozyme-treated DAP-type PG (50 ng), respectively. After 12 h, hemolymph was collected from each group, and the bactericidal effects were estimated against S. aureus and E. coli.

FIGURE 4.

The mRNA expression levels of Tenebrio and Drosophila AMPs by challenge of DAP-type PGs. A and B represent the mRNA levels of tenecin 1 and 2 in Tenebrio hemocytes (panel a) and fat bodies (panel b), respectively. C represents the mRNA levels of Drosophila drosomycin (Drs, panel a) and diptericin (Dpt, panel b) in w¹¹¹⁸ and Rel^E20 strains. Shown are insect saline (column 1), S. aureus Lys-type PG (column 2), E. coli polymeric DAP-type PG (column 3), B. subtilis polymeric DAP-type PG (column 4), and lysozyme-treated E. coli DAP-type PG (column 5). Columns 6–10 represent the same injections as those in columns 1–5 in Fig. 4C (panel a), respectively. Columns 11 and 12 represent the same injections as those in columns 1 and 3 in Fig. 4C (panel a), respectively. The mRNA levels of tenecin 1 or 2 relative to that of insect saline-injected T. molitor larvae at 12 h after injection are shown. Error bars, means ± S.D. (p ≤ 0.05) of three independent experiments.

FIGURE 5.

The amounts of Tenebrio PGRP-SA and 17-kDa protein in the hemolymph increased after injection of polymeric Lys-type and DAP-type PGs. A and B represent Western blot analysis using anti-PGRP-SA and anti-PGRP-SC2 antibodies, respectively. Lanes 1 and 2 indicate the purified PGRP-SA (500 ng) and the purified PGRP-SC2 (500 ng), respectively. Lanes 3-5, lanes 6–8, and lanes 9–11 represent the injection of insect saline (IS), monomeric DAP-type PG (mDAP), and polymeric DAP-type PG (pDAP) after 12 h, 1 days (1d), and 2 days (2d), respectively. At the indicated times, hemolymph was collected, and then a portion (40 μg of protein) of each sample was analyzed by immunoblotting using affinity-purified anti-Tenebrio PGRP-SA and PGRP-SC2 antibodies. Ab, antibody.

FIGURE 6.

Multiple amino acid sequence alignment between Tenebrio PGRP-SC2 and Drosophila PGRP family members. Three residues conserved from T7 lysozymes are shown with boxes. Two residues (His and Cys residues) that were not conserved with Tenebrio PGRP-SA are shown in boxes marked with circles. The GenBank^TM or Swissprot accession numbers for the sequences used are as follows: T. molitor (Tm) PGRP-SA, BAE78510.1; and D. melanogaster (Dm) PGRP-SC2, Q9V4X; PGRP-LB, Q9VGN3; PGRP-SB1, Q9VV97. The determined partial amino acid sequences are indicated by underlining.

FIGURE 7.

Catalytic Tenebrio PGRP-SC2 functions as a scavenger for polymeric DAP-type PG. A, kinetics of polymeric DAP-type PG degradation by Tenebrio PGRP-SC2. Insoluble polymeric DAP-type PG (1 mg/ml) was incubated with recombinant wild type PGRP-SC (5 μg/ml, ○), PGRP-SC2 (C167S)-mutant (5 μg/ml, ●), and the absence of protein (▾) in PBS (pH 7.2). B, insoluble polymeric S. aureus Lys-type PG (1 mg/ml) was incubated with different concentrations of Tenebrio recombinant PGRP-SC2, such as 0 (●), 5 (○), 10 (▾), 20 (△), and 40 (■) μg/ml. Enzymatic activity was recorded as the optical clearance of the solution at 540 nm. C, tenecin 1 (panels a and b) and tenecin 2 (panels c and d) expression were examined by injection of Tenebrio PGRP-SC2-treated polymeric DAP-type PG in the hemocytes (panels a and c) and fat bodies (panels b and d). Tenebrio larvae were challenged with insect saline (IS, column 1), polymeric DAP-type PG (12.5 μg/ml, column 2), Tenebrio PGRP-SC2-treated polymeric DAP-type PG (12.5 μg/ml, column 3), polymeric Lys-type PG only (12.5 μg/ml, column 4), and Tenebrio PGRP-SC2-treated polymeric Lys-type PG (12.5 μg/ml, column 5). Total RNA was isolated at 12 h after challenge with PGs. The mRNA levels of tenecin 1 and 2 relative to that of insect saline-injected T. molitor (Tm) larvae at 12 h after injection are shown. Error bars, means ± S.D. (p ≤ 0.05) of three independent experiments.

FIGURE 8.

Parallel comparison for the induction of AMPs after recognition of microbial molecules in beetle, fly, and mammalian. The Lys-type PG-dependent Tenebrio Toll signaling pathway was previously reported by our group (30). Drosophila Toll and Imd pathways were reviewed by Lemaitre and Hoffmann (2). The mammalian β-defensin induction pathway was reported by Ganz and co-workers (40). Namely, mammalian Toll-like receptor (TLR)-dependent expression of AMPs in keratinocytes is induced by an activated cytokine, IL-1, which is directly inducible by TLRs of monocytes and macrophages (41). A plus sign in mammalian β-defensin induction pathway means the interaction between ligand and its receptor.