Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus

Abstract

Lipoteichoic acids (LTAs) have been proposed as putative Gram-positive immunostimulatory counterparts to Gram-negative lipopolysaccharides. However, LTA from Staphylococcus aureus, the clinically most frequent Gram-positive pathogen, was inactive after purification. Here, a novel isolation procedure to prepare pure (>99%) biologically active LTA, allowing the first structural analysis by nuclear magnetic resonance and mass spectrometry, is described. A comparison with LTA purified by standard techniques revealed that alanine substituents are lost during standard purification, resulting in attenuated cytokine induction activity. In line with this finding, hydrolysis of alanine substituents of active LTA decimated cytokine induction. LTA represents a major immunostimulatory component of S. aureus.

Figure 1

TNF-α release induced by eluate fractions after HIC of a butanol extract (A) and after anion exchange rechromatography on DEAE–Sepharose of HIC-purified LTA (B) from S. aureus. Broken line (○) indicates the TNF-α concentration in picograms per milliliter of human blood, and solid line (•) displays the amount of phosphate-containing substances (including LTA). The gradients (dotted lines) indicate the increasing percentage of propanol in 0.1 M ammonium acetate buffer in the case of HIC and the increasing ammonium chloride concentration in 35% n-propanol/0.1 M ammonium acetate buffer in the case of anion exchange rechromatography, pH = 4.7, respectively.

Figure 2

(A) The structure of S. aureus LTA as determined from NMR spectroscopic analysis. Color coding identifies the methine group of the sn-glycerol repeating unit (green), three resonance signals of d-N-acetylglucosamine (GN, yellow), d-alanine (d-Ala, blue), gentiobiose-sn-glycerol (gray), and fatty acids (orange). (B) ¹H NMR spectrum (600 MHz, 300 K) of LTA obtained after butanol extraction. The resonance signal intensities allow for the quantification of the substituents and the average glycerophosphate chain length (45 < n < 50). Microheterogeneity leads to signal broadening as visible in the expansion of the anomeric proton of d-N-acetylglucosamine. (C) Hydrolysis of the d-alanine esters was performed at pH 8.5 in the NMR tube overnight. No cleavage of the fatty acids was observed under these conditions. The anomeric proton of N-acetylglucosamine resolved to a doublet with a vicinal coupling constant of 3.6 Hz as expected for an α-glycosidic bond. The gentiobiose resonances are highlighted in gray. (D) S. aureus LTA obtained after standard phenol extraction was characterized by a reduced chain length and significant hydrolysis of d-alanine esters. The alkyl chains of the fatty acids served as reference intensities for comparison of the three ¹H NMR spectra, B–D.

Figure 3

(A) Concentration dependence of TNF-α response by human whole blood to S. aureus LTA. Data are mean ± SD of three donors, 10, 100, and 1000 ng/ml LTA, significantly different (P < 0.05) from 1 ng/ml LTA and control (paired Student's t test) (B) Comparison of TNF-α induction by intact LTA (solid line, •) and dealanylated LTA fractions (broken line, ○) after HIC. Dealanylation of LTA was achieved by increasing the pH of the water phase after butanol extraction under stirring at pH ≈ 8.5 with Tris buffer at RT (21°C) for 24 h. (C) Time course of alkaline hydrolysis of intact LTA, i.e., loss in d-alanine substitution and TNF-α induction capacity.