Immune Deficiency Pathway Drosophila Allowing Detection by the Peptidoglycan Molecular Requirements

Innate immune recognition of microbes is a complex process that can be inﬂuenced by both the host and the microbe. Drosophila uses two distinct immune signaling pathways, the Toll and immune deﬁciency (Imd) pathways, to respond to different classes of microbes. The Toll pathway is predominantly activated by Gram-positive bacteria and fungi, while the Imd pathway is primarily activated by Gram-negative bacteria. Recent work has suggested that this differential activation is achieved through peptidoglycan recognition protein (PGRP)-mediated recognition of speciﬁc forms of peptidoglycan (PG). In this study, we have further analyzed the speciﬁc PG molecular requirements for Imd activation through the pattern recognition receptor PGRP-LC in both cultured cell line and in ﬂies. We found that two signatures of Gram-negative PG, the presence of diaminopimelic acid in the peptide bridge and a 1,6-anhydro form of N -acetylmuramic acid in the glycan chain, allow discrimination between Gram-negative and Gram-positive bacteria. Our results also point to a role for PG oligomerization in Imd activation, and we demonstrate that elements of both the sugar backbone and the peptide bridge of PG are required for optimum recognition. Altogether, these results indicate multiple requirements for efﬁcient PG-mediated activation of the Imd pathway and demonstrate that PG is a complex immune elicitor. The Journal of Immunology, 2004, 173: 7339–7348. (cid:4) g of PG (brieﬂy sonicated for homogeneization), and pure His 6 -tagged SltY enzyme (40 (cid:4) g) was incubated overnight at 37°C. The reaction was stopped by adding 500 (cid:4) l of 50 mM sodium phosphate buffer, pH 4.45 (HPLC eluent A), and 2 (cid:4) l of phosphoric acid. The main products generated were the monomer and dimer forms of N -acetylglucosamine (GlcNAc)-MurNAc(anhydro (anh))-tetrapeptide, but other minor tripeptide- or glycine-containing fragments were also observed that reﬂect the complexity of the PG structure, as reported by Glauner et al. (35). These different products were puriﬁed by HPLC on a Nucleosil 5C 18 column (4.6 (cid:4) 250 mm) using an isocratic elution with eluent A at 0.6 ml/min for 20 min, followed by a gradient of methanol from 0 to 25% in eluent A applied between 20 and 80 min (peaks were detected at 215 nm). Puriﬁed compounds were lyophilized, dissolved into water, and applied on the same HPLC column for desalting, using this time 0.1% triﬂuoroacetic acid and a gradient of methanol for elution. MurNAc(anh) peptides were obtained by treatment of GlcNAc-MurNAc(anh) peptides with E. coli NagZ (cid:3) N- acetylglucosaminidase. The reaction mixture (200 (cid:4) l) contained 20 mM HEPES buffer, pH 7.4, 50 mM NaCl, 0.5 mM substrate, and pure His 6 -tagged NagZ enzyme (20 (cid:4) g). GlcNAc-MurNAc(anh) tripeptides and MurNAc(anh) tripeptides were generated by treatment of the corresponding tetrapeptide compounds with E. coli LdcA L , D carboxypeptidase. The reac- His 6 -tagged LdcA enzyme (cid:4) GlcNAc-MurNAc(anh) by treatment of GlcNAc-MurNAc(anh) tetrapeptide with partially puriﬁed N -acetylmuramoyl- L -alanine amidase from E. coli . In all cases, incubations performed 37°C and products were puriﬁed desalted by HPLC, as MurNAc peptides by mild acid hydrolysis (0.1 M HCl, 10 min at 100°C) of the corresponding UDP-MurNAc peptides. Lactoyl peptides gener- ated treatment of MurNAc peptides with 4 M ammonium hydroxide 5 h at 37°C. After neutralization of the reaction mixtures with one equiv- alent of acetic acid, they were puriﬁed by HPLC, as described using triﬂuoroacetic acid as eluent. Free peptides were obtained by treat- ment of the MurNAc peptides with partially puriﬁed E. coli amidase, as Amino acid and amino sugar compositions were deter- mined with acid after hydrolysis of M HCl for 16 h at 95°C. The structure and purity of isolated PG fragments and synthesized compounds also conﬁrmed by MALDI-TOF mass spectrometry. E. SltY. SltY (cid:4) g) was incubated overnight at 37°C. The reaction was stopped by boiling during 10 min. Anhydrous products and particu-larly GlcNAc-MurNAc(anh) dipeptide were puriﬁed and then desalted by HPLC like E. coli PG products. We conﬁrmed the nature of GlcNAc-MurNAc(anh) dipeptide by MALDI-TOF mass spectrometry. MurNAc and GlcNAc residues. However, the bacterial transglycosylases catalyze an additional intramolecular transglycosylation reaction that results in the formation of a 1,6-anhydro MurNAc residue.