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

Morath S, Geyer A, Hartung T - J. Exp. Med. (2001)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacology, University of Konstanz, D-78457 Konstanz, Germany.

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.

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(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.
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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.

Mentions: The highly purified LTA induced a whole blood cytokine response at as little as 10 ng/ml (see Fig. 3 A), i.e., exhibited an immunostimulatory potency similar to that of Pseudomonas aeruginosa LPS (≥10 ng/ml was required to induce cytokine release in human whole blood); at high concentrations of 10 μg/ml LTA, TNF-α levels similar to 10 μg/ml LPS were induced.


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

Morath S, Geyer A, Hartung T - J. Exp. Med. (2001)

(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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195914&req=5

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.
Mentions: The highly purified LTA induced a whole blood cytokine response at as little as 10 ng/ml (see Fig. 3 A), i.e., exhibited an immunostimulatory potency similar to that of Pseudomonas aeruginosa LPS (≥10 ng/ml was required to induce cytokine release in human whole blood); at high concentrations of 10 μg/ml LTA, TNF-α levels similar to 10 μg/ml LPS were induced.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacology, University of Konstanz, D-78457 Konstanz, Germany.

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.

Show MeSH
Related in: MedlinePlus