Staphylococcus aureus is known to activate mammalian immune cells through Toll-like receptor 2 (TLR2).We recently demonstrated that a lipoprotein fraction obtained from S. aureus by Triton X-114 phase partitioning is a potent activator of TLR2. In this study, we separated TLR2-activating lipoproteins expressed in S. aureus and characterized an N-terminal structure. The lipoprotein fraction of S. aureus was prepared by glass bead disruption followed by Triton X-114 phase partitioning. The TLR2-activating molecules were mainly detected in the mass range of 30 -35 kDa. Seven lipoproteins were identified by the mass spectra of their tryptic digests. Among them, three lipoproteins were separated by preparative SDS-PAGE and proved to activate TLR2. After digestion with trypsin in the presence of sodium deoxycholate, the N terminus of the lipopeptide was isolated from lipoprotein SAOUHSC_02699 by normal phase high pressure liquid chromatography and characterized as an S-(diacyloxypropyl)cystein-containing peptide using tandem mass spectra. The synthetic lipopeptide counterpart also stimulated the cells via TLR2. These results showed that the diacylated lipoprotein from S. aureus acts as a TLR2 ligand in mammalian cells.Bacterial infection is one of the major causes of death. Staphylococcus aureus, the most common Gram-positive pathogen, is a major source of mortality in medical facilities (1). The pathogen causes various infectious diseases, including sepsis, endocarditis, and pneumonia. During the infection, S. aureus activates cells and evokes serious inflammation in the host. TLR2 2 has been shown to play a crucial role in the host response to S. aureus (2). However, a detailed understanding of the molecular components that interact with TLR2 in S. aureus cells has not yet been obtained. One of the reported TLR2 ligands was peptidoglycan (PGN) (3), a cell wall component of most bacteria. However, Travassos et al. (4) recently showed that PGN from several bacteria that were highly purified by removal of lipoproteins or lipoteichoic acid (LTA) were not detected by TLR2. Moreover, the minimal active components of the PGN, muramyl dipeptide and desmuramyl dipeptide (␥-D-glutamyl diaminopimelic acid), were determined to be ligands of the intracellular innate immune receptor Nod2/ Nod1 (5, 6), suggesting that PGN is not a ligand of TLR2. Another candidate of TLR2-activating ligands is LTA, a cell surface glycoconjugate of Gram-positive bacteria (3). Morath et al. (7) reported that LTA from S. aureus is a potent stimulator of cytokine release, whereas our group demonstrated that LTA from enterococci, also a major Gram-positive pathogen, has no cytokine-producing activity (8, 9). Furthermore, Han et al. (10) showed that LTA from pneumococci is 100-fold less potent than staphylococcal LTA. These observations suggested that LTA is not a common ligand of TLR2 in Gram-positive pathogens.We also found that the enterococcal LTA fraction contains some contaminants other than LTA and that these components activate immune cells t...

Lipoteichoic acid (LTA) is a cell surface glycoconjugate of gram-positive bacteria and is reported to activate the innate immune system. We previously reported that purified LTA obtained from Enterococcus hirae has no immunostimulating activity, but a subfraction (Eh-AF) in an LTA fraction possesses activity. In this study, we established a mouse monoclonal antibody neutralizing the activity of Eh-AF and investigated its inhibitory effects. Monoclonal antibody (MAbEh1) was established by the immunization of BALB/c mice with Eh-AF, followed by hybridoma screening based on its inhibitory effect for the production of interleukin-6 (IL-6) induced by Eh-AF. MAbEh1 neutralized the production of IL-6 by LTA fraction from not only E. hirae but also Staphylococcus aureus, while it failed to block that of lipopolysaccharide, suggesting that the antibody recognized a common active structure(s) in LTA fractions. Synthetic glycolipids in these LTAs did not induce cytokine production, at least in our system. Interestingly, the antibody was found to inhibit the activity of immunostimulating synthetic lipopeptides, Pam 3 CSK 4 and FSL-1. These results suggest that MAbEh1 neutralizes the activity of lipoprotein-like compounds which is responsible for the activity of the LTA fraction of E. hirae and S. aureus.Lipoteichoic acid (LTA) is a macroamphiphile distributing on the cell surfaces of gram-positive bacteria and is reported to exhibit immunostimulatory and inflammatory activities. LTA has been shown to have an antitumor effect (34, 36) and to induce inflammatory cytokines, such as tumor necrosis factor (TNF), interleukin-1 (IL-1), and 31,33). Recent research showed that such immunostimulatory activities of bacterial compounds were mediated by Toll-like receptor (TLR), a type I transmembrane receptor for innate immune activation (32). To date, more than 10 members of the TLR family have been discovered and most of their ligands were identified: TLR4 in combination with the adapter molecule MD-2 for lipopolysaccharide (LPS)/lipid A, an outer membrane component of gram-negative bacteria (21, 24); TLR9 for unmethylated CpG DNA (15); TLR3 and TLR7/8 for double-and single-stranded RNA (1, 14); TLR5 for bacterial flagellin (13); and TLR2 subfamily (TLR1, Ϫ2, and Ϫ6) for bacterial lipoprotein/lipopeptide (29, 30). LTA was also reported to be a ligand of TLR2 (22).The structures of LTAs have been well studied and proposed as a glycoconjugates generally composed of a glycolipid anchor part, such as ␤-kojibiosyldiacylglycerol for Enterococcus hirae and Streptococcus pyogenes and ␤-gentiobiosyldiacylglycerol for Staphylococcus aureus, and a 1,3-linked poly(glycerophosphate) substituted by sugars and D-alanine at position 2 of the glycerol (4). Previously, we attempted to determine a structure of the LTA responsible for these activities. Fukase et al. prepared chemically synthetic glycoconjugates having fundamental structures of LTA from E. hirae and S. pyogenes and their glycolipid anchor parts (5, 6). However, these synthetic compounds ...

Recognize this? Based on predictions from mRNA expression of lipoproteins from Staphylococcus aureus, lipopeptide fragments were chemically synthesized, and their TLR2‐stimulatory activities and cytokine‐induction capabilities were analyzed. All the lipopeptides activated TLR2, but the activities varied depending on their peptide sequences.

Acetobacter pasteurianus is an aerobic Gram-negative rod that is used in the fermentation process used to produce the traditional Japanese black rice vinegar kurozu. Previously, we found that a hydrophobic fraction derived from kurozu stimulates Toll-like receptors to produce cytokines. LPSs, particularly LPS from A. pasteurianus, are strong candidates for the immunostimulatory component of kurozu. The LPS of A. pasteurianus remains stable in acidic conditions during the 2 years of the abovementioned fermentation process. Thus, we hypothesized that its stability results from its structure. In this study, we isolated the LPS produced by A. pasteurianus NBRC 3283 bacterial cells and characterized the structure of its lipid A component. The lipid A moiety was obtained by standard weak acid hydrolysis of the LPS. However, the hydrolysis was incomplete because a certain proportion of the LPS contained acid-stable D-glycero-D-talo-oct-2-ulosonic acid (Ko) residues instead of the acid-labile 3-deoxy-D-manno-oct-2-ulosonic acid residues that are normally found in typical LPS. Even so, we obtained a Ko-substituted lipid A with a novel sugar backbone, ␣-Man(1-4)[␣-Ko(2-6)]␤-GlcN3N(1-6)␣-GlcN(1-1)␣-GlcA. Its reducing end GlcN(1-1)GlcA bond was also found to be quite acid-stable. Six fatty acids were attached to the backbone. Both the whole LPS and the lipid A moiety induced TNF-␣ production in murine cells via Toll-like receptor 4, although their activity was weaker than those of Escherichia coli LPS and lipid A. These results suggest that the structurally atypical A. pasteurianus lipid A found in this study remains stable and, hence, retains its immunostimulatory activity during acetic acid fermentation.

No abstract