Structural decomposition and heterogeneity of commercial lipoteichoic Acid preparations

Abstract

Fractionation of commercial preparations of lipoteichoic acids (LTA) by hydrophobic interaction chromatography (HIC) and nuclear magnetic resonance spectroscopy revealed very inhomogeneous compositions and decomposition of the LTA structure: LTA content of the preparations averaged 61% for Streptococcus pyogenes, 16% for Bacillus subtilis, and 75% for Staphylococcus aureus. The decomposition was characterized by a loss of glycerophosphate units as well as alanine and N-acetylglucosamine substituents. All preparations contained-to varying degrees-non-LTA, non-lipopolysaccharide (LPS) immunostimulatory components as indicated by their elution profile in HIC, lack of phosphate, and negative Limulus amoebocyte lysate (LAL) test results. After purification, the commercial LTA from Bacillus subtilis and S. pyogenes but not LTA from S. aureus induced the release of tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), IL-6, and IL-10 in human blood. While pure LTA are negative in the LAL assay, endotoxin equivalents of more than 10 ng of LPS/mg of LTA were found in the commercial preparations. Taken together, these data indicate that these crude preparations with relatively high endotoxin contamination are not suitable for characterizing the activation of immune cells by LTA.

FIG. 1.

Elution profile of phosphate content and TNF-α stimulatory activity after HIC purification of commercial LTA from S. aureus and B. subtilis. Twenty-five milligrams of commercial LTA preparations from S. aureus (Sigma, lot 86H4085) (A) or B. subtilis (Sigma, lot 17H4021) (B) were subjected to HIC. Fractions were analyzed for phosphate content (•), indicative of LTA in hydrophobic fractions, and for their TNF-α stimulatory activity (○) by incubating 1 ml of 20% blood in the presence of 10 μl of eluate for 24 h. ▴, TNF-α release of unstimulated blood; ▾, TNF-α release induced by 100 ng of LPS/ml.

FIG. 2.

Concentration-dependent induction of TNF-α in human whole blood by commercial, commercial HIC-purified, and butanol-extracted LTAs. Twenty percent whole blood from three donors was incubated in the presence of the concentrations of different LTA indicated for 24 h. TNF-α was measured in the cell supernatant by ELISA. Commercial LTA from B. subtilis and LTA from S. aureus were purified by HIC, pooling the phosphate-containing hydrophobic fractions. The start material was compared to this preparation as well as a butanol-extracted LTA from the same species prepared in our laboratory.

FIG. 3.

A-D ¹H NMR spectra of commercial and butanol-extracted LTA preparations. The ¹H NMR spectra of LTA from S. aureus (butanol-extracted) (A), LTA from S. aureus (Sigma) (B), LTA from B. subtilis (butanol extracted) (C), or LTA from B. subtilis (Sigma) (D) are shown.

FIG. 4.

Structure of B. subtilis LTA. The structure was deduced from the NMR analysis of butanol-extracted LTA from B. subtilis shown in Fig. 3.