Isolation of small peptidoglycan fragments from the cell wall of Staphylococcus aureus. Cell walls were purified as described in the Methods. They are represented as follows (top left corner): open circle, N-acetylglucosamine; open square, N-acetylmuramic acid; closed circles, amino-acid residues of the stem peptides; red ovals, proteins associated with the cell wall; blue ovals, teichoic acids. Cell walls were treated with trypsin to degrade associated proteins and incubated in 48% HF at 4°C to cleave the phosphodiester bonds between the repeating units of the teichoic acids and the linkage to PG. The resulting PG was digested with mutanolysin to produce the constituent muropeptides, which were then separated by HPLC in monomers (muropeptides 5), dimers (muropeptide 11), trimers (muropeptide 15), tetramers (muropeptide 16) and higher polymers (top HPLC chromatogram). A schematic representation of their chemical structure is shown along with the respective chromatograms (see Methods for details). Electron microscopy photograph was kindly provided by Dr M.G. Pinho (ITQB, Portugal).

Reduction of the free end in the sugar chain renders muropeptides inactive. (A) Dimeric muropeptide of Staphylococcus aureus. Muropeptides are present as anomers representing three states of MurNAc. The aldehyde group of the C1 of the muramic acid residue is converted to the respective alditol after reduction, resulting in a molecule unable to circularize. Conversely, as lysostaphin converts dimers to monomers (B), these changes abolish elicitor activity as seen with the tetrameric and dimeric muropeptides of S. aureus. No induction of drs was observed in dif¹ and PGRP^seml mutants.

Polymerized muropeptides from Gram-positive peptidoglycan activate the Toll pathway. (A) A dimeric GlcNAc–MurNAc is the minimum structure required for Staphylococcus aureus (Sa) PG fragments to induce drs expression. Polymerization augments expression levels, as seen with the injection of trimeric or tetrameric muropeptides. To compare drs expression levels induced by small PG fragments of S. aureus, we used as positive controls cell wall and PG from Micrococcus luteus (Ml), a well-documented inducer of the Toll pathway (Werner et al, 2000; Michel et al, 2001; Leulier et al, 2003). All compounds were used at a concentration of 0.15 mg/ml unless otherwise stated. Cell wall and PG were used at 5 mg/ml. (B) Purified components of the pneumonococcal cell wall were tested for their elicitor activity. PG from Streptococcus pneumoniae (S pneu) induces drs expression at levels comparable with injection of cell wall from the same bacterium. Although monomeric muropeptides do not activate drs, dimers induce a response albeit in lower levels compared with PG. Conversely, polymeric (poly) muramyl peptides show a stronger activation. These polymeric muropeptides represent fractions following the dimer in HPLC-mediated purification. All compounds were used at a concentration of 0.1 mg/ml unless otherwise stated. PG samples were used at 5 mg/ml. Values for (A,B) are mean values of five independent experiments with the relevant standard deviation.

PGRPsA but not GNBP1 mediates recognition of muropeptides. (A) Both GNBP1^osiris and PGRP-SA^seml mutants fail to respond to PG from Micrococcus luteus (Ml PG) or Staphylococcus aureus (Sa PG). Nevertheless, only flies deficient for PGRP-SA are unsuccessful in responding to purified muropeptides. (B) When Ml PG is mixed with haemolymph of wild-type flies at 37°C, hydrolysis occurs (WT). On the contrary, mixing with haemolymph from GNBP1^osiris mutant flies does not result in PG hydrolysis (osiris). Bringing back GNBP1 (through a UAS–GNBP1 construct driven by yolk GAL4) in an osi background restores activity. PG incubated with mutanolysin (M) was used as a control for PG hydrolysis. Values shown are mean values from three independent experiments with the relevant standard deviation.