Prophenoloxidase activation and antimicrobial peptide expression induced by the recombinant microbe binding protein of Manduca sexta

Abstract

Manduca sexta microbe binding protein (MBP) is a member of the β-1,3-glucanase-related protein superfamily that includes Gram-negative bacteria-binding proteins (GNBPs), β-1,3-glucan recognition proteins (βGRPs), and β-1,3-glucanases. Our previous and current studies showed that the purified MBP from baculovirus-infected insect cells had stimulated prophenoloxidase (proPO) activation in the hemolymph of naïve and immune challenged larvae and that supplementation of the exogenous MBP and peptidoglycans (PGs) had caused synergistic increases in PO activity. To explore the underlying mechanism, we separated by SDS-PAGE naïve and induced larval plasma treated with buffer or MBP and detected on immunoblots changes in intensity and/or mobility of hemolymph (serine) proteases [HP14, HP21, HP6, HP8, proPO-activating proteases (PAPs) 1-3] and their homologs (SPH1, SPH2). In a nickel pull-down assay, we observed association of MBP with proHP14 (slightly), βGRP2, PG recognition protein-1 (PGRP1, indirectly), SPH1, SPH2, and proPO2. Further experiments indicated that diaminopimelic acid (DAP) or Lys PG, MBP, PGRP1, and proHP14 together trigger the proPO activation system in a Ca²⁺-dependent manner. Injection of the recombinant MBP into the 5th instar naïve larvae significantly induced the expression of several antimicrobial peptide genes, revealing a possible link between HP14 and immune signal transduction. Together, these results suggest that the recognition of Gram-negative or -positive bacteria via their PGs induces the melanization and Toll pathways in M. sexta.

Keywords: Hemolymph protease system; Insect immunity; Lys- or DAP-type peptidoglycan; Melanization; Pattern recognition; Toll pathway.

Fig. 1. Increase of proPO activation in induced hemolymph caused by recombinant M. sexta MBP in the absence (A) or presence (B) of microbial elicitor

(A) As described in Section 2.3, aliquots of diluted cell-free hemolymph from immune challenged larvae were incubated with 0 to 100 ng (left panel) and 50 to 1000 ng (right panel) MBP or BSA. PO activities are shown as mean ± SEM (n = 3). The concentration of endogenous MBP in the mixture was estimated to be 24 to 32 nM, using known amounts of the purified recombinant MBP as standards running side-by-side with the plasma samples on SDS-PAGE followed by immunoblot with MBP antibodies. (B) 5 μl of 1:10 diluted plasma from induced larvae were incubated with buffer (#1), 20 ng purified MBP (#2), 1 μg elicitor (E. coli soluble DAP-PG, B. mageterium insoluble DAP-PG, M. luteus insoluble Lys-PG, or S. aureus soluble LTA, #3), or elicitor and MBP both (#4) prior to PO activity measurement. Since interaction of plasma factors with elicitor (#2 − #1) and co-presence of plasma and exogenous MBP (#3 − #1) both lead to proPO activation, an interaction of elicitor and exogenous MBP in plasma (#4 − #1) is anticipated to increase proPO activation to a level significantly higher than the sum of the two components [i.e. (#2 − #1) + (#3 − #1)]. Therefore, the PO activity changes represented by (#4 − #1) and (#2 + #3 − 2×#1), or simply #4 and (#2 + #3 − #1), are compared using Student’s t-test. An asterisk (*) indicates that #4 is significantly higher than #2 + #3 − #1 (p < 0.05), supporting a synergistic effect of the elicitor-MBP interaction.

Fig. 2. Detection of changes in intensity and mobility of hemolymph proteins in control (NP) and induced (IP) plasma from M. sexta larvae

As described in Section 2.4, NP or IP was incubated with buffer (lane 1 for NP, lane 3 for IP) or MBP (lane 2 for NP, lane 4 for IP). Following SDS-PAGE and electrotransfer, protein blots were separately incubated with diluted polyclonal antisera, as indicated in the bottom of each panel. The eight M_r marker proteins are shown as short bars with their sizes indicated on the left. Positions of the precursor proteins (arrowheads), cleavage products (arrow for catalytic/protease-like domain and circle for regulatory chain), putative serine protease-serpin complexes (asterisk), and PO oligomers (diamond) are indicated. Some of the cross-reactive bands are unclear.

Fig. 3. Hemolymph proteins associated with MBP

As described in Section 2.5, induced plasma was incubated with BSA (lane 1) or MBP (lane 2) for 1 h at 4°C before affinity chromatography. Bound proteins were eluted, aliquoted, and separated by 10, 12 or 15% SDS-PAGE, and subjected to immunoblot analysis using specific polyclonal antibodies indicated in the bottom of each panel. Each lane is equivalent to the bound proteins from 7.5 μl of the IP. Positions of the M_r markers are shown as short bars with their sizes marked. Intact protein, arrowhead; protease-like domain, arrow; regulatory chain, circle. The 55 kDa band (marked ●) crossreacted by the diluted βGRP2 and PGRP1 antiserum is MBP. In lane 3 of the last panel, the binding to MBP was performed in the presence of E. coli DAP-PG.

Fig. 4. Ca²⁺-dependent activation of M. sexta proHP14 by PGs recognized by MBP and PGRP1 and HP14-induced proPAP2 activation via HP21

As described in Section 2.6, the purified MBP, PGRP1, proHP14, E. coli PG, and buffer were incubated at 37°C for 90 min, along with the control reactions. The reaction mixtures were treated with 1×SDS sample buffer in the presence (A and first two lanes of B) or absence (last two lanes of B) of DTT, separated by 10% SDS-PAGE, and subjected to immunoblot analysis using HP14 antiserum as the primary antibody. EDTA at a final concentration of 10 mM was included in the reaction to test whether or not Ca²⁺ chelation blocks proHP14 cleavage activation (C). Lys-PG from S. aureus (D: right lane) or DAP-PG from E. coli (D: middle lane) was incubated with proHP14, MBP, and PGRP1 to test possible difference in the extent of proHP14 auto-proteolysis. Like the laminarin-βGRP2 binary complex, the ternary complex of DAP-PG, MBP and PGRP1 caused proHP14 auto-proteolysis, HP14 activated proHP21, HP21 activated proPAP2, and PAP2 hydrolyzed IEARpNA (E).

Fig. 5. Up regulation of antimicrobial protein genes by injection of MBP

As described in Section 2.7, buffer or MBP was injected into larvae and fat body was dissected 24 h later for total RNA isolation, cDNA synthesis, and qPCR analysis. Relative abundances of AMP transcripts were calculated based on average Ct values from two technical repeats and plotted as a bar graph (mean ± SEM).

Fig. 6

A model for proHP14 auto-proteolysis and HP14-triggered activation of the SP-SPH system in M. sexta.