Comparison of Lipoteichoic Acid from Different Serotypes of Streptococcus pneumoniae

Abstract

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that ofStaphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae: R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitolbound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon ␥). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.Streptococcus pneumoniae is one of the most common Gram-positive pathogens that colonizes the upper respiratory tract and causes many severe infections like otitis media, sinusitis, and more life-threatening diseases like pneumonia, bacteremia, and meningitis, when it gains access to the lower respiratory tract or the bloodstream (1, 2). In the United States alone, there were in the last 20 years approximately 7 million cases of otitis media each year, 500,000 cases of pneumonia, 50,000 cases of bacteremia, and 3,000 cases of meningitis (3, 4). S. pneumoniae also causes a high mortality rate of 40,000 per year in the United States. When the infection has cleared, patients often retain neurological sequelae like hearing impairment or learning disabilities.Most bacteria are surrounded by a capsule, which makes recognition by the immune system more difficult. On the basis of the differences in composition of the capsular polysaccharides, S. pneumoniae can be divided into Ͼ90 serotypes (5, 6). But, only seven serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) are responsible for 65% of all cases of pneumococcal disease (7) and 23 serotypes for 90% (8).A prevalent problem with S. pneumoniae infections is the emergence of antibiotic-resistant strains in the last years. Previous studies show an increase in penicillin resistance of S. pneumoniae from 4% to 21%...

Streptococcus pneumoniae Serotype 11D Has a Bispecific Glycosyltransferase and Expresses Two Different Capsular Polysaccharide Repeating Units

“…This conclusion was further supported by the presence of another weak signal at 4.02/54.47 ppm, which arises from an H2 containing geminal N-acetylation (Fig. 3A) and is distinct from the 4.32/50.0 ppm and 4.10/51.3 ppm signals assigned to ␣-N-acetylgalactosamine (␣GalNAc) H2 and ␤GalNAc H2, respectively, in pneumococcal teichoic acid (cwPS) (15). A diffusion-ordered spectroscopy experiment (16) revealed no evidence of free ␣GlcNAc in the CPS sample solution.…”

Lipoteichoic acid from an Enterococcus faecalis clinical strain promotes TNF-α expression through the NF-κB and p38 MAPK signaling pathways in differentiated THP-1 macrophages

“…The n-butanol method and HIC on octyl-sepharose were used to extract and purify LTA. The butanol method can be used to extract structurally intact LTA and has been widely used to extract LTA from numerous types of Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus pneumoniae, E. faecalis and Streptococcus gordonii (6,19,23,24). Furthermore, SDS-PAGE and Coomassie Blue G-250 staining were used to detect protein contamination.…”

“…The structure of the LTA was assayed by 1 H nuclear magnetic resonance (NMR) spectrometry as described previously (19). The purity of the LTA was determined by measuring the protein content through Coomassie Blue G-250 staining (Bio-Rad, Hercules, CA, USA) following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gradient gels containing 5-12% wt/vol acrylamide (Bio-Rad).…”

“…The processes of isolation and purification of LTA were performed as described previously (19). The bacteria were defrosted and suspended in 100 ml of 0.1 M cold sodium citrate buffer (pH 4.7) (Sigma-Aldrich).…”

“…Endotoxin contamination was excluded using a Limulus amoebocyte lysate (LAL) assay kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Thus, the endotoxin was quantified and the contamination was evaluated (19,20). DNA or RNA contamination was determined by measuring ultraviolet (UV) absorption at 260 and 280 nm (NanoDrop Spectrophotometer 2000; Thermo Fisher Scientific, Wilmington, DE, USA) (21).…”