Peptidoglycan recognition protein-SD provides versatility of receptor formation in Drosophila immunity

Abstract

In Drosophila, the enzymatic activity of the glucan binding protein GNBP1 is needed to present Gram-positive peptidoglycan (PG) to peptidoglycan recognition protein SA (PGRP-SA). However, an additional PGRP (PGRP-SD) has been proposed to play a partially redundant role with GNBP1 and PGRP-SA. To reconcile the genetic results with events at the molecular level, we investigated how PGRP-SD participates in the sensing of Gram-positive bacteria. PGRP-SD enhanced the binding of GNBP1 to Gram-positive PG. PGRP-SD interacted with GNBP1 and enhanced the interaction between GNBP1 and PGRP-SA. A complex containing all three proteins could be detected in native gels in the presence of PG. In solution, addition of a highly purified PG fragment induced the occurrence not only of the ternary complex but also of dimeric subcomplexes. These results indicate that the interplay between the binding affinities of different PGRPs provides sufficient flexibility for the recognition of the highly diverse Gram-positive PG.

Fig. 1.

Production of functional recombinant PGRP-SD in insect cells. (A) Purified PGRP-SD (predicted molecular mass: 20 kDa) was analyzed on a 15% SDS-reducing gel, indicating a single protein species. Protein was detected by Coomassie blue staining. Further identification by N-terminal sequencing (EVPIVT) showed a mature protein with the N-terminal signal peptide cleaved. (B) PGRP-SD^Δ3 mutant flies are highly susceptible to S. aureus infection compared with WT adults. Injecting 20 ng of rPGRP-SD before infection, however, rescued lethality, producing a survival pattern comparable to that in WT animals. Survival patterns were plotted using Kaplan–Meier analysis. Statistical comparisons (log-rank test) were as follows: WT vs. SD rescue, P = 0.256; SD rescue vs. PGRP-SD^J3, P < 0.0001; WT vs. PGRP-SD^J3, P < 0.0001.

Fig. 2.

Enhanced binding of PGRP-SA and GNBP1 to PG and cell wall in the presence of PGRP-SD. (A) 20 μg of PGRP-SA were incubated with PG from Ml (top panel), Sa (middle panel), and Ss (lower panel) (see Materials and Methods) with or without PGRP-SD. Note that the 2 bands observed in lane c (Sa panel) are both PGRP-SA with and without the 6xHis tag. In our hands, storage of rPRGP-SA results in partial loss of the tag (L.W., unpublished observations). The identity of the bands was confirmed by an Ab against PGRP-SA (marks both bands) and one against the His tag (marks the upper band solely; data not shown). (B) 20 μg of GNBP1 were incubated with PG as above in the presence or absence of PGRP-SD. GNBP1 binding was significantly enhanced by PGRP-SD in a molecular ratio of 1:1 (or 20-μg GNBP1 and 10 μg of PGRP-SD) for all cases (lanes b). All of the protein bands in SDS-PAGE gel were visualized by Coomassie blue (CB) staining. We verified the presence of PGRP-SD by using an antibody against the protein in western blots (WB).

Fig. 3.

PGRP-SD interacts with both PGRP-SA and GNBP1. (A) rPGRP-SD was immobilized on a CM5 sensor chip, and serial dilutions of rGNBP1 were injected. The RU increased in accordance with the increasing concentration of the injected protein. (B) PGRP-SD stabilized the interaction between GNBP1 and PGRP-SA. rPGRP-SA was immobilized onto the CM5 sensor chip. rGNBP1 and rPGRP-SD were mixed at a 1:1 molar ratio and injected with a serial dilution from 18.5 μM to 0.144 μM. (C) The binding kinetics of all experiments (those described above and all reciprocal orientations) are presented. Ligand refers to the protein coated on the sensor chip. The term analyte is used for the protein injected.

Fig. 4.

Ternary complex formation in the presence of PG. Processed PG shifted all of the three proteins into a higher-molecular-weight complex in native Tris-glycine PAGE gels. 6 μg of rPGRP-SA, rPGRP-SD, and rGNBP1 were used in single protein species controls (lanes 1–3). rGNBP1 mixed with either rPGRP-SA or rPGRP-SD as a 1:2 molar ratio is shown in lanes 4 and 5. The rPGRP-SA, rPGRP-SD, and rGNBP1 mix at 2:2:1 ratio is shown in lane 6. PG from Ml processed by rGNBP1 as described during 12 h (lane 7), 24 h (lane 8), or 48 h (lane 9) was incubated with the 3-protein mix (as defined in lane 6). Protein mix incubated with PG digested completely by mutanolysin (24 h) was used as control (lane 10). Arrows illustrate potential protein complexes. Lower bands in lanes 8, 9, and 10 represent PG fragments bound to PGRP-SA. In this form PGRP-SA will be running faster than the loading control of protein alone because AUC data indicate that it becomes more spherical upon microbial ligand addition (see Table S1, where S value increases from 2.0 to 2.2 upon muropeptide addition). Protein bands were visualized by Coomassie blue staining.

Fig. 5.

PGRP-SD, PGRPSA, and GNBP1 during host receptor signaling in vivo. (A) Kaplan–Meier analysis of survival experiments after Sa infection using double or single mutants of host receptors were carried out. Survival tests were performed in three independent experiments. Log-rank tests comparing the three experiments for each genotype indicated that the experiments were homogeneous (all P values very high and in the range of 0.7–0.98). Thus, it was valid to pool these experiments and for each genotype to analyze the data for all flies used. Because there were 35 pairwise tests between different genotypes there was an issue of multiplicity of tests. To address that, we used the Bonferroni correction for multiple tests (0.05 divided by the number of tests). This placed P values for significance at or <0.0014. The log-rank tests for the comparisons of the 8 genotypes presented in the graph gave P values that clearly placed the WT and Dif flies in one group (WT vs. Dif, P = 0.214), the single mutants in a second group, and the double mutants in a third group, whereby the statistical difference between all single vs. double mutant tests was significant (P < 0.0005) as judged by: GNBP1 vs. GNBP1, PGRP-SD, P < 0.0005; GNBP1 vs. PGRP-SA; GNBP1, P < 0.0005; PGRP-SA vs. PGRP-SA; PGRP-SD, P < 0.0005; PGRP-SA vs. PGRP-SA; GNBP1, P < 0.0005; PGRP-SD vs. PGRP-SD, GNBP1, P < 0.0005; and PGRP-SD vs. PGRP-SA; PGRP-SD, P < 0.0005. All single or double mutant combinations vs. the WT gave P < 0.0005. Differences between the single mutants were not statistically significant (GNBP1 vs. PGRP-SA, P = 0.003; GNBP1 vs. PGRP-SD, P = 0.033; PGRP-SA vs. PGRP-SD, P = 0.428). Finally, differences between double mutants were also not statistically significant (GNBP1, PGRP-SD vs. PGRP-SA; GNBP1, P = 0.008; GNBP1, PGRP-SD vs. PGRP-SA; PGRP-SD, P = 0.007; and PGRP-SA; GNBP1 vs. PGRP-SA; PGRP-SD, P = 0.512). (B) Concomitant overexpression of PGRP-SA and GNBP1 through the GAL4/UAS system results in activation of the AMP gene drosomycin (drs) used as a read-out for activation of the Toll pathway. This activation occurs in the absence of any immune challenge and amounts to 40% of drs induction after infection. Expressing PGRP-SD through a UAS-transgene at the same time as PGRP-SA and GNBP1 induces drs at the level seen by Gram-positive bacterial infection (S. aureus). Columns represent the percentage mean value of three independent experiments (corrected against the loading control RP49) with standard deviation represented as error bars. Asterisks indicate values that are not statistically different (P > 0.005) from each other. All other differences are statistically significant (P < 0.005).