Andreas Stadelmaier

Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.

We recently purified lipoteichoic acid (LTA) from Staphylococcus aureus to more than 99% purity by a novel preparation method and deduced its structure with the first nuclear magnetic resonance (NMR) of a complete LTA. In contrast to Gram-negative lipopolysaccharides, this LTA requires the toll-like receptor (TLR)-2 and not TLR-4 for cytokine induction in monocytes and macrophages. To elucidate the structure–function relationships for LTA from S. aureus, the lipid anchor was prepared by either acidic hydrolysis of native LTA or chemical synthesis (gentiobiosyl-sn-dimyristoylglycerol). Next, a complete LTA molecule with six glycerophosphate units carrying four alanine plus one N-acetyl-glucosamine substituent was synthesized, which displayed the same potency to activate monocytes as native LTA. However, 100–1,000 times higher concentrations of the lipid anchor were required for cytokine induction. It is worthy to note that replacing d-alanine with l-alanine blunted the effect indicating stereoselective recognition. The structure identification of this synthesized and biologically active LTA was proven by NMR and matrix-assisted laser desorption-ionization mass spectrometry. We concluded that the lipid anchor, with its fatty acids, represents an integral part of the immunostimulatory activity of LTA, but requires additional structural components on the polyglycerophosphate backbone.

Definition of Structural Prerequisites for Lipoteichoic Acid-Inducible Cytokine Induction by Synthetic Derivatives

The controversy about the immune stimulatory properties of lipoteichoic acid (LTA) from Staphylococcus aureus was solved recently by showing decomposition and inactivation of LTA obtained by conventional purification strategies, as well as pronounced LPS contamination of commercial preparations. By introducing a novel preparation method, the structure of bioactive LTA was elucidated. This structure was confirmed by chemical synthesis. In this work, synthetic LTA derivatives were employed to study the structure-function relationship of cytokine induction in human monocytes. Synthetic LTA induced the same cytokine pattern as highly purified natural LTA. The gentiobiose core could be omitted without affecting bioactivity. The polyglycerophosphate backbone amplified the response to the lipid anchor (∼100-fold) only when substituted with d-alanine, whereas α-d-N-acetylglucosamine substituents could be omitted. Replacing d-alanine substituents with l-alanine reduced the activity of the molecule at least 10-fold, indicating stereoselectivity. These results define for the first time the crucial patterns required for the immune recognition of LTA.

Use of Synthetic Derivatives To Determine the Minimal Active Structure of Cytokine-Inducing Lipoteichoic Acid

ABSTRACT Lipoteichoic acid (LTA) from gram-positive bacteria is the counterpart to lipopolysaccharide from gram-negative bacteria. LTA, which activates Toll-like receptor 2 (TLR2), induces a unique cytokine and chemokine pattern. The chemical synthesis of LTA proved its immunostimulatory properties. To determine the minimal active structure of LTA, we reduced synthetic LTA in a number of steps down to the synthetic anchor and employed these molecules to stimulate interleukin-8 (IL-8) release in human whole blood. Ten times more of the synthetic structures with four to six d-alanine-substituted polyglycerophosphate units (50 nM) than of the native LTA preparation was required to induce IL-8 release. A further reduction to three backbone units with two or no d-alanine residues resulted in cytokine induction only from 500 nM. The synthetic anchor was not able to induce IL-8 release even at 5 μM. When the LTA derivatives were used at 500 nM, they induced increasing levels of IL-8 and tumor necrosis factor alpha with increasing elongation of the backbone. Peritoneal macrophages were less responsive than human blood to the synthetic structures. Therefore, TLR2 dependency could be shown only with cells from TLR2-deficient mice for the two largest synthetic structures. This was confirmed by using TLR2-transfected HEK 293 cells. Taken together, these data indicate that although the synthetic anchor (which, unlike the native anchor, contains only myristic acid) cannot induce cytokine release, the addition of three backbone units, even without d-alanine substituents, confers this ability. Lengthening of the chain with d-alanine-substituted backbone units results in increased cytokine-inducing potency and a more sensitive response.