Innate immune sensing of Staphylococcus aureus unravels basic mechanisms leading to either effective antibacterial immune responses or harmful inflammation. The nature and properties of S. aureus-derived pathogen-associated molecular pattern (PAMPs) are still not completely understood. We investigated the innate immune sensing of peptidoglycan (PGN) structures and subsequent immune consequences. Macromolecular PGN (PGN(polymer)) preparations activated NF-κB through human Toll-like receptors 2 (TLR2), as shown by luciferase reporter assays, and induced murine dendritic cell (DC) maturation and cytokine production. In contrast, PGN(polymer) from lgt-mutant S. aureus failed to stimulate human TLR2, demonstrating that lipoproteins within the macromolecular structures of PGN(polymer), but not PGN itself, activate TLR2. Thus, HPLC-purified monomeric PGN (PGN(monomer)) structures were investigated. Strikingly, PGN(monomer) completely lacked NF-κB activation, lacked TLR2 activity, and failed to functionally activate murine DCs. However, PGN(monomer) in concert with various TLR ligands most effectively stimulated DCs to up-regulate IL-12p70 and IL-23 by ≥3- to 5-fold. Consequently, DCs coactivated by PGN(monomer) markedly up-regulated Th1 and Th17 while suppressing Th2 cell priming. Notably, PGN(monomer) failed to coactivate NOD2(-/-) DCs. This demonstrates that PGN(monomer) is a natural ligand of NOD2, which was previously only demonstrated for synthetic compounds like muramyl dipeptide. Interestingly, murine DCs lacking TLR2 remained mute in response to the combinative immune sensing of S. aureus-derived PAMPs, including PGN(monomer), providing for the first time an explanation of why S. aureus can colonize the nasal mucosa in the absence of inflammation. This is very likely based on the lack of TLR2 expression in mucosal epithelial cells under normal conditions, which determines the unresponsiveness to S. aureus PAMPs.

The capability of FT-IR transmission spectrometry was examined for the direct determination of glucose in whole blood without any sample preparation. For these investigations, the whole blood samples were automatically aspirated by a syringe pump into the transmission cell. Infrared spectra were recorded in the 1500–900 cm−1 range. Despite the high water background absorption and the complex blood matrix, significant spectral changes due to different glucose concentrations were observed. Chemometric (partial least-squares) models were applied for the determination of glucose. A standard error of calibration of 13.8 mg/dL was obtained by using a partial least-squares calibration model containing five ranks. The residues for an independent test set were less than 15 mg/dL.

Peptidoglycan muropeptides, potent proinflammatory components, are amidated in Staphylococcus aureus for unknown reasons. To study whether this modification may modulate proinflammatory capacity, cytokine induction by isogenic S. aureus strains with different amidation levels and by synthetic amidated/nonamidated muramyldipeptides was evaluated. However, amidation did not significantly affect cytokine induction. This finding contributes to defining peptidoglycan receptor specificities and indicates that further rationales for muropeptide amidation have to be considered.Staphylococcus aureus is a frequent constituent of human nasal microflora and a major cause of severe endogenous infections (11). The staphylococcal cell wall determines several key aspects of the infection process, such as adherence (11, 28), immune recognition (13), and resistance to host defenses (21,22). While many adhesive surface proteins have been investigated (12), the structure, function, and variability of nonproteinaceous polymers, such as peptidoglycan (PG) or teichoic acids, have remained parts of a neglected field of research. S. aureus is known to modify the canonical PG structure by O acetylation of the glycan strand and by amidation of the ␣-carboxyl group of the D-glutamate (D-Glu) residue in muropeptides, resulting in the formation of D-isoglutamine (16, 29). While O acetylation confers lysozyme resistance (3), the primary role of D-Glu amidation has remained unclear. Nevertheless, the latter modification is known to affect the level of methicillin resistance (4, 17) and to contribute to vancomycin susceptibility in S. aureus (8). The amino group for D-Glu amidation in S. aureus muropeptides is most probably derived from free glutamine (17, 23). However, the responsible transamidase enzyme has not yet been identified. Gustafson et al. described a femC mutant with 48% decreased muropeptide amidation (17, 26). The femC mutant has a disrupted glnR gene encoding the regulator of the glutamine synthetase (Fig. 1A), which results in reduced glutamine synthetase activity. As a consequence of lower amounts of the amino group donor, muropeptide amidation is reduced in this mutant (17).PG has potent proinflammatory properties (5). It is sensed by the human innate immune system via NOD1 or NOD2 proteins (15,18). An additional role of the TLR2 receptor in PG sensing has been described previously (10) but remains a matter of debate (27). The minimal structure required for NOD1 recognition is a dipeptide consisting of D-Glu and meso-diaminopimelate (mesoDAP), which is mainly produced by gram-negative bacteria (14, 15). D-Glu amidation (leading to D-isoglutamine) strongly impairs PG recognition by NOD1 (7), and the amidation of mesoDAP completely abrogates the ability of NOD1 to detect PG (15). NOD2 has a different substrate specificity as it requires a muramyldipeptide (MDP) composed of N-acetylmuramic acid, L-alanine, and D-Glu (Fig. 1B) (19,27). S. aureus PG is recognized by NOD2 but not by NOD1 since it contains L-lysine instead of meso-D...

Two strains, Pseudomonas aeruginosa TB10839 and TB121838, which belong to the TB clonal lineage, have been isolated from sputa of cystic fibrosis patients. Despite the fact that the strains are closely related, their pathogenic potential differs dramatically: while strain TB10839 is capable of proliferating in polymorphonuclear granulocytes, strain TB121838 is not. Comparative two-dimensional polyacrylamide gel electrophoresis coupled to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry was employed to map the extracellular, intracellular, and surface sub-proteomes of TB10839 and TB121838 and to identify differentially expressed proteins. About 4% of all detected protein spots were differentially expressed between both strains including absent or present spots and spots with a more than 2-fold changed intensity. This percentage reflects a relatively high degree of intraclonal variability. Many of the protein spots in TB10839 that were missing or expressed at lower levels in TB121838 were identified as quorum-sensing regulated virulence factors. It might be speculated that the increased expression of these proteins contributes to pathogenic competence of TB10839.

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