Evidence of Immunostimulating Lipoprotein Existing in the Natural Lipoteichoic Acid Fraction

Abstract

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 ...

Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis

“…Our data are in support of those of Hashimoto et al (15)(16)(17) who reported that contaminating BLPs carry the immunostimulatory activity commonly assigned to LTA from S. aureus. Thus, in contrast to LTA, GBS BLPs qualify as highly potent bacterial toxins.…”

“…However, the LTA response was amplified only by muramyl dipeptides (MDP), the minimal PGN fragments that interact with the intracellular NOD receptors, and not by macromolecular PGN (5,13,14). In disagreement with this model, however, recent work from Hashimoto and colleagues (15)(16)(17) challenged LTA from S. aureus as a TLR2 agonist altogether. Hence, we were left with the puzzle of whether LTA might act in concert with other GBS substructures (e.g., MDP) in vitro and in vivo or whether molecules from GBS other than LTA were more potent stimulants of TLR2.…”

Internalization and Coreceptor Expression Are Critical for TLR2-Mediated Recognition of Lipoteichoic Acid in Human Peripheral Blood

“…The latter authors demonstrated a 100-fold decreased immunostimulatory capacity of LTA preparations derived from a Dlgt deletion mutant S. aureus lacking palmitate-labeled lipoproteins compared with LTA from the respective wild-type (wt-LTA) strain. From this finding and from other observations, they concluded contaminating lipoproteins in the LTA preparations to be responsible for LTA-mediated immune activation (24)(25)(26). Contrary to the latter investigators (27), we found that LTA of a mutant S. aureus strain lacking lipoproteins (Dlgt-LTA) and wt-LTA were equipotent in inducing cytokine release from human whole blood.…”

“…Despite strong evidence for a major role of LTA in the immune activation by Gram-positive bacteria (21)(22)(23), recent reports suggested that not LTA but lipoproteins are dominant immunobiologically active structures of S. aureus (24,25). The latter authors demonstrated a 100-fold decreased immunostimulatory capacity of LTA preparations derived from a Dlgt deletion mutant S. aureus lacking palmitate-labeled lipoproteins compared with LTA from the respective wild-type (wt-LTA) strain.…”

Application of hybrid LRR technique to protein crystallization

“…Bacterial lipoproteins are anchored to bacterial membrane through three lipid chains covalently attached to the conserved N-terminal cysteine (37). Lipoteichoic acids are amphipathic molecules found in the membrane of Gram positive bacteria and induce strong proinflammatory signals in macrophages (38)(39)(40).…”