A Short-Type Peptidoglycan Recognition Protein 1 (PGRP1) Is Involved in the Immune Response in Asian Corn Borer, Ostrinia furnacalis (Guenée)

Abstract

The insect immune response is initiated by the recognition of invading microorganisms. Peptidoglycan recognition proteins (PGRPs) function primarily as pattern recognition receptors by specifically binding to peptidoglycans expressed on microbial surfaces. We cloned a full-length cDNA for a PGRP from the Asian corn borer Ostrinia furnacalis (Guenée) and designated it as PGRP1. PGRP1 mRNA was mainly detected in the fat bodies and hemocytes. Its transcript levels increased significantly upon bacterial and fungal challenges. Purified recombinant PGRP1 exhibited binding activity to the gram-positive Micrococcus luteus, gram-negative Escherichia coli, entomopathogenic fungi Beauveria bassiana, and yeast Pichia pastoris. The binding further induced their agglutination. Additionally, PGRP1 preferred to bind to Lys-type peptidoglycans rather than DAP-type peptidoglycans. The addition of recombinant PGRP1 to O. furnacalis plasma resulted in a significant increase in phenoloxidase activity. The injection of recombinant PGRP1 into larvae led to a significantly increased expression of several antimicrobial peptide genes. Taken together, our results suggest that O. furnacalis PGRP1 potentially recognizes the invading microbes and is involved in the immune response in O. furnacalis.

Keywords: Ostrinia furnacalis (Guenée); agglutination; binding; innate immune response; peptidoglycan recognition proteins (PGRPs); prophenoloxidase stimulation.

Figure 1

Sequence alignment of O. furnacalis PGRP1 with PGRPs from other insects. The GenBank accession numbers for the sequences used are as follows: DmPGRP-SA, Q9VYX7; PGRP-SD, Q9VS97; PGRP-SC1, Q9V3B7; PGRP-SC2, Q9V4X2; PGRP-LB, Q9VGN3; BmPGRP-S1, NM_001043371; MsPGRP1, AF413068; Lysozyme, NP_041973. The predicted signal peptides are shaded in gray. The conserved Cys residues for disulfide bonds are indicated in red. Residues involved in the binding pocket in DmPGRP-SA and -LB are labeled in red boxes. Residues essential for T7 lysozyme activity are shaded in blue. The putative peptidoglycan-recognition positions are shaded in yellow. Secondary structures are assigned the above sequences from the N to C termini of amino acid sequences.

Figure 2

Expression profile analysis of O. furnacalis PGRP1. (A) Expression profiles of O. furnacalis PGRP1 at different stages of development. RNA was extracted from the whole bodies of collected eggs; first-instar (L1), second-instar (L2), third-instar (L3), fourth-instar (L4), and fifth-instar (L5) larvae; and pupae. The rpL8 gene was used as an internal control. (B) Expression profiles of O. furnacalis PGRP1 in various tissues. Hd, head; Mg, midgut; Hc, hemocytes; Fb, fat body (O. furnacalis). (C) Expression profiles of O. furnacalis PGRP1 upon microbial challenge. On day 0, fifth-instar larvae were infected with PBS (Control), E. coli, M. luteus, or B. bassiana. RNA was prepared from the whole bodies 24 h after injection. The rpL8 gene was used as an internal standard to indicate a consistent total mRNA amount. Bars labeled with different letters are significantly different (one–way ANOVA, followed by Tukey’s multiple comparisons test, p < 0.05).

Figure 3

SDS-PAGE (A) and immunoblot (B) analysis of purified PGRP1. The purified recombinant PGRP1 was separated by 15% SDS-PAGE followed by Coomassie Brilliant Blue staining (lane 1, 3 μg) or immunoblotting (lane 2, 0.6 μg) with mouse monoclonal anti-polyhistidines as primary antibodies. The sizes and positions of the molecular weight standards are indicated on the right.

Figure 4

Binding of recombinant PGRP1 to different microorganisms (A) and peptidoglycans (B). (A) Recombinant PGRP1 was incubated with E. coli, M. luteus, B. bassiana, and P. pastoris. After incubation for 10 min, the incubated mixtures were pelleted and washed three times, and the pellet was resuspended. The samples from each step were separated by 15% SDS-PAGE and visualized by immunoblotting with mouse monoclonal anti-polyhistidine as the primary antibody. Lane 1, recombinant PGRP1 (0.1 μg); lane 2, total mixture before centrifugation; lane 3, the supernatant after centrifugation (unbound fraction); lane 4, the fraction after washing three times; lane 5, the suspended pellet (bound fraction). (B) Binding of PGRP1 to peptidoglycans. Microtiter plates were coated by DAP-type peptidoglycan from E. coli K12 or Lys-type peptidoglycan from M. luteus. Increasing concentrations of rPGRP1 were added to the coated wells (the same amount of rGFP was added as a control). The binding assay was performed as described in “Materials and methods”. Each point represents the mean ± S.D. (n = 3). The solid lines represent the nonlinear regression calculation of the one-site binding curve.

Figure 5

Agglutination of microorganisms by recombinant PGRP1. Different concentrations of recombinant PGRP1 (100 μg/mL, 150 μg/mL, and 200 μg/mL) were incubated with acridine orange-stained E. coli (2 × 10⁸ cells/mL), M. luteus (2 × 10⁸ cells/mL), or B. bassiana (1 × 10⁵ conidia/mL), respectively, in PBS for 45 min at room temperature. BSA (200 μg/mL) instead of rPGRP1 was used as a control. The agglutination of microbial cells was then observed under invert fluorescence microscopy.

Figure 6

Stimulation of PPO activation in O. furnacalis plasma upon the addition of recombinant PGRP1. Recombinant PGRP1 (100 μg/mL) or rGFP (as a control) were mixed with plasma from O. furnacali fifth-instar day 0 larvae and incubated in the presence or absence of E. coli (1.6 × 10⁸ cells/mL) at room temperature for 10 min. Details of the reaction mixtures are provided in the figures. The bars represent mean ± S.D. (n = 3). Bars labeled with different letters are significantly different (one–way ANOVA, followed by Tukey’s multiple comparisons test, p < 0.05).

Figure 7

(A) Schematic diagram to explain how the injection of PGRP1 and B. bassiana boosted the expression of AMPs. The recognition and binding of O. furnacalis PGRP1 to PAMP in B. bassiana stimulated the activation of serine protease cascades. In one pathway, it resulted in the conversion of PPO into PO, whose activity increased greatly. In the other pathway, it led to the activation of pro-Spätzle, which further bound to the Toll receptor to initiate the expression of AMP genes, including attacin, cecropin-4, gloverin-1, moricin-4, etc. (B) Induced expression of AMP genes in O. furnacalis upon the injection of recombinant PGRP1. Purified recombinant PGRP1 or GFP (2 μg) were injected into the day 0 fifth-instar larvae of O. furnacalis. For some treatments, the larvae were injected again 30 min later with B. bassiana conidia suspension (5 × 10³ conidia) or PBS as a control. Total RNA samples were isolated from the whole body at 24h after injection for qRT-PCR analysis. O. furnacalis rpL 8 was used as an internal standard to normalize the templates. The bars represent the mean ± S.D. (n = 3). Bars labeled with different letters are significantly different (analysis using one-way ANOVA followed by Newman–Keuls test, p < 0.05).