International Immunology

International Immunology Advance Access originally published online on December 22, 2005
International Immunology 2006 18(2):355-362; doi:10.1093/intimm/dxh374

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 18/2/355    most recent dxh374v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (14) Request Permissions Google Scholar Articles by Hashimoto, M. Articles by Suda, Y. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, M. Articles by Suda, Y. Social Bookmarking          What's this?

© The Japanese Society for Immunology. 2005. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Lipoprotein is a predominant Toll-like receptor 2 ligand in Staphylococcus aureus cell wall components

Masahito Hashimoto¹, Kazuki Tawaratsumida¹, Hiroyuki Kariya¹, Kazue Aoyama², Toshihide Tamura² and Yasuo Suda¹

¹ Department of Nanostructure and Advanced Materials, Kagoshima University, Korimoto 1-21-40, Kagoshima 890-0065, Japan
² Department of Microbiology, Hyogo College of Medicine, Mukogawa 1-1, Nishinomiya 663-8501, Japan

Correspondence to: M. Hashimoto; E-mail: hassy{at}eng.kagoshima-u.ac.jp

	Abstract

Top Abstract Introduction Methods Results and Discussion References

 
Lipoteichoic acid (LTA) derived from Staphylococcus aureus is reported to be a ligand of Toll-like receptor 2 (TLR2). In this study, we demonstrated that lipoproteins obtained from S. aureus are potent activators of TLR2. A fraction obtained by Triton X-114 phase partitioning activated cells through TLR2. The fraction contained proteins and LTA. The activity was detected in compounds in a mass range of 12–40 kDa. Proteinase K digested the active compounds into lower molecular weight active materials <10 kDa. In contrast, hydrofluoric acid treatment, which decomposes LTA, did not alter the molecular mass of the active compounds. Further, most of the activity was abrogated by lipoprotein lipase digestion. These results suggested that lipoproteins are predominant TLR2 ligands in S. aureus cell wall components.

Keywords: innate immunity, lipoteichoic acid, PAMPs, TLR, Triton X-114

	Introduction

Top Abstract Introduction Methods Results and Discussion References

 
The innate immune system plays key roles in host defense against bacterial infection. The host recognizes bacterial components known as pathogen-associated molecular patterns (PAMPs) and regulates cellular responses. Toll-like receptor (TLR), a type I transmembrane protein, has been found to act as a major signaling receptor for PAMPs (1). To date, >10 members of the TLR family have been discovered and most of their ligands were identified. TLR4, the most characterized member of the family, in combination with an adapter molecule MD-2 has been shown to recognize LPS, an outer membrane component of gram-negative bacteria (2, 3). TLR9 has been reported to be involved in immune responses to unmethylated CpG DNA (4), and TLR3 and TLR7/8 sense viral double- and single-stranded RNA (5, 6). Activation of TLR5 has been demonstrated to be mediated by bacterial flagellin (7). Bacterial lipoproteins have been found to be stimuli of TLR2 subfamily (TLR1, 2 and 6) (8, 9). TLR recognizes PAMPs through an extracellular leucine-rich repeat domain and activates signal transduction cascades via a cytoplasmic Toll/IL-1 receptor domain (10). The cascades involving MyD88, IRAK, TAK1 and TRAF6 activate NF-B, and lead to expression of inflammatory mediator genes.

Bacterial infection is one of the major causes of death. Staphylococcus aureus, a most common gram-positive pathogen, is a major source of mortality in medical facilities (11). The pathogen causes various infectious diseases, including sepsis, endocarditis and pneumonia. During the infection, S. aureus activates cells and evokes serious inflammation in the host. TLR2 has been shown to play a crucial role in the host response to S. aureus (12). However, detailed information on molecular components interacting with TLR2 in S. aureus cells is still unclear. Peptidoglycan (PGN), a cell wall component of most bacteria, was reported to be a TLR2 ligand (13). However, Travassos et al. (14) recently showed that PGN from several bacteria, which are highly purified by removal of lipoprotein or lipoteichoic acid (LTA), are not sensed through TLR2. Moreover, the active minimal components in PGN, muramyl dipeptide and desmuramyl dipeptide, were determined to be ligands of intracellular innate immune receptor Nod1/Nod2 (15–18), indicating that PGN is not a ligand of TLR2. LTA, a cell surface glycoconjugate of gram-positive bacteria, has been considered to be a candidate for a TLR2-activating ligand (13). Morath et al. (19) reported that LTA from S. aureus was a potent stimulus of cytokine release. Whereas, we recently demonstrated that LTA from enterococci, also a major gram-positive pathogen, has no cytokine-producing activity (20, 21). Further, Han et al. (22) showed that LTA from pneumococci is 100-fold less potent than staphylococcal LTA. These observations suggested that LTA is not a common ligand of TLR2 in gram-positive pathogen.

We previously found that enterococcal LTA fraction contains some contaminants other than LTA and the components activate immune cells through TLR2 (20, 23). However, we have not yet identified their structures essential for the activity. TLR2 is known to be a predominant receptor utilized by lipoproteins derived from various bacteria. Inflammatory response by membrane lipoproteins from gram-negative bacteria, such as Borrelia burgdorferi, Escherichia coli, Neisseria gonorrhoeae and Porphyromonas gingivalis are mediated by TLR2 (24–27). Mycoplasmal lipoproteins, which are from Mycoplasma fermentans and Mycoplasma salivarium, also activate innate immunity via TLR2 (28–31). Further their N-terminal lipopeptides, which contained di or triacylated S-(2,3-dihydroxypropyl)cystein, have been proven as essential moieties for the activity using chemically synthesized compounds. Although lipoproteins are known to exist also in gram-positive bacterial cell wall (32), it remains unknown whether the lipoproteins in gram-positive bacteria exhibit inflammatory activities via TLR2. In the present study, we extracted lipoproteins from S. aureus and characterized their immunobiological activities.

	Methods

Top Abstract Introduction Methods Results and Discussion References

 
Bacterial culture and extraction of cell wall components
Staphylococcus aureus DSM 20231 organisms were grown in nutrient broth (Becton Dickinson, Franklin Lakes, NJ) at 37°C for 6 h with constant shaking. The bacterial cells were harvested by centrifugation, then washed three times with saline and lyophilized. To extract a fraction containing lipoproteins, S. aureus cells were subjected to Triton X-114 (TX-114) phase partitioning according to the method as reported (33). Briefly, the cells were suspended in PBS containing protease inhibitor cocktail Complete mini (Roche Diagnostics, Basel, Switzerland) and combined with one-tenth volume of 10% aqueous TX-114. The mixture was rotated at 4°C for 1 h and then cell debris were centrifuged off. The supernatant was incubated and centrifuged at 37°C to separate TX-114 from aqueous phase. The upper aqueous phase was treated again with TX-114. Crude lipoprotein fraction, designated as Sa-TX (Scheme 1A), was precipitated from TX-114 phase by addition of excess methanol.

View larger version (17K): [in this window] [in a new window]   Scheme 1. Separation procedure for cell wall components from S. aureus.

 
Crude LTA faction of S aureus was prepared by aqueous n-butanol (BuOH) extraction method (19). Briefly, the cells were suspended in BuOH/water (1:1, v/v) and the mixture was stirred at room temperature for 30 min and centrifuged to separate aqueous phase from BuOH and cell debris. The aqueous phase was dialyzed at 4°C and lyophilized to give crude LTA fraction, designated as Sa-Bu (Scheme 1B).

Chemical and enzymatic degradation
Sa-TX was dissolved in 40 mM octylglucoside in PBS and digested with 20 ng µl^–1 trypsin (Promega, Madison, WI) or 50 ng µl^–1 proteinase K (Takara Bio, Shiga, Japan) at 37°C for 16 h. Sa-TX was also treated with aqueous 47% hydrofluoric acid (HF) at 4°C for 16 h and then lyophilized over sodium hydroxide. The HF hydrolysate was again subjected to TX-114 phase partitioning as described above to give lipoprotein fraction, designated as Sa-LP (Scheme 1A). Sa-LP dissolved in octylglucoside was digested with 50 ng µl^–1 lipoprotein lipase (Sigma-Aldrich, St Louis, MO) at 37°C for 16 h. Sa-Bu was also subjected to HF treatment as described above. The lyophilized HF hydrolysate was washed twice with chloroform–methanol (2/1, v/v) to remove glycolipid anchor from LTA, and then subjected to lipoprotein lipase digestion in octylglucoside solution.

Analytical methods
Phosphorous contents were measured by the method of Bartlett (34). Contamination of LPS was detected by Limulus amebocyte lysate assay using Endospecy® test (Seikagaku, Tokyo, Japan), a Limulus clotting factor C-specific quantitative chromogenic assay reagent. SDS-PAGE was performed by the Tris–glycine method (35) using a mini PAGE chamber AE-6530 and an AE-8450 power supply (ATTO, Tokyo, Japan) with a 15% gel. Proteinous materials were visualized by silver (Ag) or Coomassie brilliant blue (CBB) staining and acidic materials, such as LTA, by Alcian blue staining. Monocyte western blotting was carried out by the method as previously described (36). Briefly, stimuli were separated by SDS-PAGE, and the resolved stimuli in the gel were transblotted to a nitrocellulose membrane by the Towbin method (37) using an AE-6677 semi-dry blotting apparatus (ATTO). The membrane of each lane was cut into 4-mm strips and each strip was separately dissolved in dimethyl sulfoxide. The solution was poured into PBS to precipitate stimuli-coated particles which consist of stimuli and nitrocellulose. After washing three times with PBS, the particle suspension in assay medium was applied to a luciferase assay using Ba/TLR2 cells described below.

Luciferase assays
Ba/F3 cells stably expressing p55IgLuc, an NF-B/DNA binding activity-dependent luciferase reporter construct (Ba/B), murine TLR2 and the p55IgLuc reporter construct (Ba/mTLR2) and murine TLR4/MD-2 and the p55IgLuc reporter construct (Ba/mTLR4/mMD-2) were kindly provided by Prof. K. Miyake (Institute of Medical Science, University of Tokyo, Japan). NF-B-dependent luciferase activity in these cells was determined as described previously (27). Briefly, cells were inoculated onto each well of a 96-well flat bottomed plate (Becton Dickinson) at 1 x 10⁵ cells in 80 µl of RPMI1640 (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) (MBL, Nagoya, Japan), and stimulated with the indicated doses of the test specimens. After 4 h incubation at 37°C in humidified air containing 5% CO₂, 80 µl of Bright-Glo^TM luciferase assay reagent (Promega) was added to each well and luminescence was quantified with a luminometer ARVO SX multilabel counter (Perkin Elmer, Wellesley, MA). Results are shown as relative luciferase activity, which was the ratio of stimulated activity to non-stimulated activity, in each cell line.

Cytokine assay
A mouse macrophage cell line, J774A.1 (Health Science Research Resource Bank, Osaka, Japan) was cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS. Cells were plated onto 96-well flat bottomed plate at 2 x 10⁵ cells per well and stimulated with the indicated doses of the test specimens in culture medium supplemented with or without 1% FBS for 4 h at 37°C. After incubation, culture supernatants were collected and used for the cytokine assay using an ELISA kit for secreted tumor necrosis factor (TNF)- (R&D systems). The concentration of secreted TNF- from cells was determined using a standard curve of recombinant TNF- prepared in each assay.

mRNA expression
Eight-week-old male BALB/c mice were obtained from Kyudo (Kumamoto, Japan). The animals received humane care in accordance with our institutional guidelines and the legal requirements of Japan. Elicited peritoneal macrophages were obtained from mice 3 days after intraperitoneal inoculation of 1.0 ml of 3% sterile Brewer's thioglycolate broth (Becton Dickinson). Peritoneal exudate cells were centrifuged and suspended in RPMI1640 supplemented with 10% FBS. These cells were then distributed to 3.5-cm dish at 2.5 x 10⁶ cells ml^–1, after which they were incubated for 2 h at 37°C in humidified air containing 5% CO₂. Each dish was washed twice with PBS to remove non-adherent cells, and those attached to the dish served as peritoneal macrophages. Cells were stimulated with the indicated doses of the test specimens in culture medium supplemented with 1% FBS for 1.5 h at 37°C. Total RNA was extracted by a single-step extraction method with TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription (RT)–PCR was performed by two-step method using an RNA PCR kit (Takara Bio). RT of extracted total RNA to cDNA was performed with oligo-dT primer and PCR was carried out with sense and antisense oligonucleotide primers specific for mouse TNF-, IL-1ß, IL-6 or ß-actin (38) using a GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, CA). For a negative control, a non-RT sample was amplified by PCR. PCR products were detected by electrophoresis on a 1.5% agarose gel. Real-time PCR was performed with the cDNA by a SYBR® Premix Ex Taq kit (Takara Bio) and primer pairs purchased from Takara Bio using an ABI PRISM® 7700 (Applied Biosystems).

	Results and Discussion

Top Abstract Introduction Methods Results and Discussion References

 
Staphylococcus aureus TX-114 extract activates cells via TLR2
According to the previous report that TX-114 phase partitioning extracts lipoproteins from bacterial cells (31), S. aureus cells were treated with TX-114 to give crude lipoprotein fraction, Sa-TX, which yielded 0.1% from dried cells (Scheme 1A). Sa-TX stimulated induction of TNF- production in J774A.1 (Fig. 1A). Sa-TX also induced NF-B activation in Ba/mTLR2 cells, but not in Ba/mTLR4/mMD-2 and Ba/B (Fig. 1B). These results indicated that the cell activation by Sa-TX is mediated by TLR2. SDS-PAGE profile of Sa-TX showed that Sa-TX contained Alcian blue-positive acidic compounds in addition to various kinds of Ag- and/or CBB-positive component (Fig. 2B), suggesting that TX-114 treatment extracts LTA as well as specific proteins from whole bacterial proteins (Fig. 2A). Since LTA has been reported to be a ligand for TLR2 (39, 40), we extracted an LTA fraction from S. aureus cells by BuOH extraction (Sa-Bu, 1.0% yield, Scheme 1B) and compared their activities. In the SDS-PAGE profile of Sa-Bu (Fig. 2C), a smear band was visualized by Alcian blue staining at the molecular mass range similar to that of Sa-TX, showing the existence of LTA in the fraction. However, no visible band was detected in Ag and CBB staining gels, suggesting that the amount of contaminated protein is under detection limit or the protein is poorly stained. Although Sa-Bu also induced TNF- production (Fig. 1A) and NF-B activation via TLR2 (Fig. 1B), the activity was slightly less potent than Sa-TX. LTA contents of the fractions were estimated by phosphorous analysis. Sa-Bu contained 1.67 µmol/mg phosphorous, while Sa-TX contained 0.35 µmol/mg, suggesting that LTA content in Sa-TX is one-fifth of that in Sa-Bu. Further no Limulus clotting factor C activation was observed in Sa-TX up to 10 µg ml^–1, indicating no endotoxin contamination.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Immunobiological activity of the extracts from Staphylococcus aureus bacteria. (A) TNF- production induced by Sa-TX and Sa-Bu in J774A.1 cells. (B) NF-B activation induced by Sa-TX (500 ng ml–1) and Sa-Bu (500 ng ml–1) in Ba/B, Ba/mTLR2 and Ba/mTLR4/mMD-2 cells.

 

View larger version (47K): [in this window] [in a new window]   Fig. 2. SDS-PAGE profiles of (A) Staphylococcus aureus whole lysate, (B) Sa-TX and (C) Sa-Bu. The gels were visualized by Ag, CBB or Alcian blue (AB).

 
LTA is not responsible for stimulation by Sa-TX
We next examined the active component in Sa-TX. Sa-TX was separated by SDS-PAGE and analyzed by monocyte western blotting. By the analysis, TLR2-mediated NF-B activation was detected in a molecular mass range of 10–40 kDa (Fig. 3A). In contrast, LTA visualized by Alcian blue staining was found in the range of 12–20 kDa (Fig. 3A). In SDS-PAGE profiles of proteinase K-digested Sa-TX, most of CBB-positive bands disappeared, while NF-B activation was retained but detected in the area of <10 kDa (Fig. 3B). However, the LTA band was still displayed in the same range (Fig. 3B). Similar results were obtained by using trypsin-digested Sa-TX (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Separation of TLR2-activating components in (A) Sa-TX, (B) proteinase K-digested Sa-TX and (C) HF-treated Sa-TX. SDS-PAGE gels were visualized by CBB or Alcian blue (AB). NF-B activation was detected by monocyte western blotting using Ba/mTLR2 cells.

 
We further investigated the effect of HF degradation of Sa-TX. Since HF cleaves the phosphodiester bond, polyglycerophosphate, a hydrophilic part of LTA and most of molecular mass of LTA is decomposed into small components, such as phosphate, glycerol and phosphoglycerol (41). After HF degradation no Alcian blue-stained band was found in the SDS-PAGE gel, whereas most of CBB-positive bands in the gel still remained. NF-B activity in monocyte western analysis was observed in the range of 10–35 kDa (Fig. 3C). Although the activity of high molecular mass materials was reduced probably due to hydrolysis in the acidic condition, most of the activity was retained. These observations suggested that proteinous materials rather than LTA are predominant immunostimulatory compounds in Sa-TX.

Lipoproteins stimulate cytokine production in immune cells
Sa-TX was subjected to HF degradation and subsequent TX-114 phase partitioning to give a lipoprotein-rich fraction, Sa-LP (Scheme 1B). TLR2-stimulating activity of Sa-LP was slightly decreased compared with that of Sa-TX but retained (Fig. 4A). This slight reduction might be caused by degradation of LTA or hydrolysis of active components in an acidic condition. In contrast, NF-B activation through TLR2 in Sa-LP was abrogated by lipoprotein lipase digestion (Fig. 4A). The activity of lipoproteins was reported to be largely reduced (42). Thus, the results indicated that active compounds in Sa-LP are lipoproteins. In addition to NF-B activation, Sa-LP activated J774A.1 to induce TNF- production (Fig. 5A) and murine peritoneal macrophages to express inflammatory cytokine genes, TNF-, IL-1ß and IL-6 (Fig. 5B) in a similar manner to LPS. mRNA expression levels were also determined by real-time PCR (Fig. 5C–E). These observations showed that Sa-LP strongly activates innate immune systems though it is 10- to 100-fold less potent than LPS.

View larger version (26K): [in this window] [in a new window]   Fig. 4. NF-B activation in Ba/mTLR2 cells. (A) Sa-TX, Sa-LP (HF-treated Sa-TX), lipoprotein lipase-digested Sa-LP (LPL Sa-LP) and lipoprotein lipase (LPL) as negative controls. (B) Sa-Bu, HF-treated Sa-Bu (HF Sa-Bu) and lipoprotein lipase-digested HF-treated Sa-Bu (LPL HF Sa-Bu). (C) Delipidated HF-treated Sa-Bu (dHF Sa-Bu) and lipoprotein lipase-digested delipidated HF-treated Sa-Bu (LPL dHF Sa-Bu). (D) SDS-PAGE profiles of Sa-Bu and HF-treated Sa-Bu (HF Sa-Bu).

 

View larger version (39K): [in this window] [in a new window]   Fig. 5. Proinflammatory cytokine production induced by Sa-LP. (A) TNF- production in J774A.1. Expression of mRNA induced by Sa-LP and LPS in murine peritoneal macrophages. (B) PCR products visualized by gel electrophoresis. Relative mRNA expression determined by real-time PCR for (C) TNF-, (D) IL-1ß and (E) IL-6.

 
Similarly Sa-Bu was subjected to HF degradation. After HF degradation, no Alcian blue-stained band was found in the SDS-PAGE gel of Sa-Bu (Fig. 4D). However, TLR2-stimulating activity of HF-treated Sa-Bu was not abrogated (Fig. 4B), indicating that Sa-Bu also contained HF-resistant active compounds. Further, HF-treated Sa-Bu was subjected to delipidation followed by lipoprotein lipase digestion. As shown in Fig. 4(C), TLR2-dependent activity was reduced by digestion, suggesting the existence of lipoprotein in Sa-Bu. It is worth noting that direct lipoprotein lipase digestion of HF-treated Sa-Bu (Fig. 4B) as well as Sa-Bu (data not shown) was unsuccessful. It might be caused by micelle formed by LTA or glycolipid anchor from hydrolyzed LTA, which hides lipid region of lipoprotein from enzyme.

LPS is a well-known TLR4 ligand. But commercial enterobacterial LPS preparations have been shown to slightly induce a signal via TLR2, in addition to TLR4. The activity is shown to be the result of contaminants in these preparations, which can be removed by phenol re-extraction (43) and were identified as lipoproteins (25). It has been reported that non-enterobacterial LPS preparations also exhibit activities via TLR2 even after re-extraction (44, 45). However, the component responsible for the activity was also identified as a contaminated lipoprotein in the fraction (27, 46). These studies suggested that the identification of the PAMPs from bacterial components is difficult, because perfect removal of contamination from the objective target molecule, especially for high molecular mass heterogeneous glycoconjugates, such as LPS or LTA, may be impossible. In this study, we demonstrated that TLR2-stimulating active compounds in Sa-LP are lipoproteins. We also indicated that Sa-Bu contained lipoproteins. Morath et al. (19) extracted an LTA fraction by BuOH method and separated by hydrophobic interaction followed by anion exchange chromatography. In their elution profiles, cytokine-inducing activity was eluted as a single peak. However, our results suggested a possibility that the fractions of LTA and lipoprotein come together. Thus, lipoprotein contamination in LTA obtained from natural sources should be further investigated.

Recently, Stoll et al. (47) constructed a lipoprotein diacylglycerol transferase (lgt) deletion mutant of S. aureus. The mutant completely lacked palmitate-labeled lipoproteins and the cells and crude lysate induced less proinflammatory cytokines than wild type. These reports further support our conclusion that lipoproteins are the predominant TLR2 ligand in S. aureus. In the S. aureus genome, >50 putative lipoproteins were identified (47). Although we have not yet identified proteins responsible for the activities, at least several kinds of such lipoproteins are considered to be expressed in the cells and extracted in Sa-LP.

In conclusion, we demonstrated here that lipoproteins obtained from S. aureus are stimuli for innate immune system and the activation is mediated via TLR2. Our finding supported the importance of TLR2 for inflammation evoked by S. aureus.

	Acknowledgements

 
This work was supported in part by Grants-in-Aid for Encouragement of Young Scientists (B) (16710160) from the Ministry of Education, Culture, Sports, Science and Technology and for Scientific Research (C) (17510179) from the Japanese Society of the Promotion of Science. We thank Prof. Kazuhisa Sugimura at Kagoshima University for measuring luciferase activities.

	Abbreviations

 

Ag	silver
BuOH	n-butanol
CBB	Coomassie brilliant blue
HF	hydrofluoric acid
LTA	lipoteichoic acid
PAMPs	pathogen-associated molecular patterns
PGN	peptidoglycan
RT	reverse transcription
TLR	Toll-like receptor
TNF	tumor necrosis factor
TX-114	Triton X-114

	Notes

 
Transmitting editor: M. Miyasaka

Received 24 August 2005, accepted 22 November 2005.

	References

Top Abstract Introduction Methods Results and Discussion References

 

1. Underhill, D. M. and Ozinsky, A. 2002. Toll-like receptors: key mediators of microbe detection. Curr. Opin. Immunol. 14:10.
2. Poltorak, A., He, X., Smirnova, I. et al. 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085.[Abstract/Free Full Text]
3. Shimazu, R., Akashi, S., Ogata, H. et al. 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J. Exp. Med. 189:1777.[Abstract/Free Full Text]
4. Hemmi, H., Takeuchi, O., Kawai, T. et al. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740.[CrossRef][Medline]
5. Alexopoulou, L., Holt, A. C., Medzhitov, R. and Flavell, R. A. 2001. Recognition of double-stranded RNA and activation of NF-B by Toll-like receptor 3. Nature 413:732.[CrossRef][Medline]
6. Heil, F., Hemmi, H., Hochrein, H. et al. 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526.[Abstract/Free Full Text]
7. Hayashi, F., Smith, K. D., Ozinsky, A. et al. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410:1099.[CrossRef][Medline]
8. Takeuchi, O., Kawai, T., Muhlradt, P. F. et al. 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int. Immunol. 13:933.[Abstract/Free Full Text]
9. Takeuchi, O., Sato, S., Horiuchi, T. et al. 2002. Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins. J. Immunol. 169:10.[Abstract/Free Full Text]
10. Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1:135.[CrossRef][Medline]
11. Lowy, F. D. 1998. Staphylococcus aureus infections. N. Engl. J. Med. 339:520.[Free Full Text]
12. Takeuchi, O., Hoshino, K., Kawai, T. et al. 1999. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity 11:443.[CrossRef][Web of Science][Medline]
13. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M. and Kirschning, C. J. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like receptor 2. J. Biol. Chem. 274:17406.[Abstract/Free Full Text]
14. Travassos, L. H., Girardin, S. E., Philpott, D. J. et al. 2004. Toll-like receptor 2-dependent bacterial sensing does not occur via peptidoglycan recognition. EMBO Rep. 5:1000.[CrossRef][Web of Science][Medline]
15. Inohara, N., Ogura, Y., Fontalba, A. et al. 2003. Host recognition of bacterial muramyl dipeptide mediated through NOD2. Implications for Crohn's disease. J. Biol. Chem. 278:5509.[Abstract/Free Full Text]
16. Girardin, S. E., Boneca, I. G., Viala, J. et al. 2003. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. 278:8869.[Abstract/Free Full Text]
17. Girardin, S. E., Boneca, I. G., Carneiro, L. A. et al. 2003. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 300:1584.[Abstract/Free Full Text]
18. Chamaillard, M., Hashimoto, M., Horie, Y. et al. 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat. Immunol. 4:702.[CrossRef][Web of Science][Medline]
19. Morath, S., Geyer, A. and Hartung, T. 2001. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J. Exp. Med. 193:393.[Abstract/Free Full Text]
20. Suda, Y., Tochio, H., Kawano, K. et al. 1995. Cytokine-inducing glycolipids in the lipoteichoic acid fraction from Enterococcus hirae ATCC 9790. FEMS Immunol. Med. Microbiol. 12:97.[Medline]
21. Hashimoto, M., Yasuoka, J., Suda, Y. et al. 1997. Structural feature of the major but not cytokine-inducing molecular species of lipoteichoic acid. J. Biochem. 121:779.[Abstract/Free Full Text]
22. Han, S. H., Kim, J. H., Martin, M., Michalek, S. M. and Nahm, M. H. 2003. Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcal LTA in stimulating Toll-like receptor 2. Infect. Immun. 71:5541.[Abstract/Free Full Text]
23. Hashimoto, M., Imamura, Y., Morichika, T. et al. 2000. Cytokine-inducing macromolecular glycolipids from Enterococcus hirae: improved method for separation and analysis of its effects on cellular activation. Biochem. Biophys. Res. Commun. 273:164.[CrossRef][Web of Science][Medline]
24. Hirschfeld, M., Kirschning, C. J., Schwandner, R. et al. 1999. Cutting edge: inflammatory signaling by Borrelia burgdorferi lipoproteins is mediated by Toll-like receptor 2. J. Immunol. 163:2382.[Abstract/Free Full Text]
25. Lee, H. K., Lee, J. and Tobias, P. S. 2002. Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling. J. Immunol. 168:4012.[Abstract/Free Full Text]
26. Fisette, P. L., Ram, S., Andersen, J. M., Guo, W. and Ingalls, R. R. 2003. The Lip lipoprotein from Neisseria gonorrhoeae stimulates cytokine release and NF-B activation in epithelial cells in a Toll-like receptor 2-dependent manner. J. Biol. Chem. 278:46252.[Abstract/Free Full Text]
27. Hashimoto, M., Asai, Y. and Ogawa, T. 2004. Separation and structural analysis of lipoprotein in a lipopolysaccharide preparation from Porphyromonas gingivalis. Int. Immunol. 16:1431.[Abstract/Free Full Text]
28. Takeuchi, O., Kaufmann, A., Grote, K. et al. 2000. Cutting edge: preferentially the R-stereoisomer of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 activates immune cells through a Toll-like receptor 2- and MyD88-dependent signaling pathway. J. Immunol. 164:554.[Abstract/Free Full Text]
29. Ozinsky, A., Underhill, D. M., Fontenot, J. D. et al. 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc. Natl Acad. Sci. USA 97:13766.[Abstract/Free Full Text]
30. Nishiguchi, M., Matsumoto, M., Takao, T. et al. 2001. Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by Toll-like receptor 2: role of N-terminal hydrophobic portion in its multiple functions. J. Immunol. 166:2610.[Abstract/Free Full Text]
31. Fujita, M., Into, T., Yasuda, M. et al. 2003. Involvement of leucine residues at positions 107, 112, and 115 in a leucine-rich repeat motif of human Toll-like receptor 2 in the recognition of diacylated lipoproteins and lipopeptides and Staphylococcus aureus peptidoglycans. J. Immunol. 171:3675.[Abstract/Free Full Text]
32. Sutcliffe, I. C. and Russell, R. R. 1995. Lipoproteins of gram-positive bacteria. J. Bacteriol. 177:1123.[Free Full Text]
33. Shibata, K., Hasebe, A., Sasaki, T. and Watanabe, T. 1997. Mycoplasma salivarium induces interleukin-6 and interleukin-8 in human gingival fibroblasts. FEMS Immunol. Med. Microbiol. 19:275.[CrossRef][Web of Science][Medline]
34. Bartlett, G. R. 1959. Phosphorus assay in column chromatography. J. Biol. Chem. 234:466.[Free Full Text]
35. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[CrossRef][Medline]
36. Wallis, R. S., Amir-Tahmasseb, M. and Ellner, J. J. 1990. Induction of interleukin 1 and tumor necrosis factor by mycobacterial proteins: the monocyte western blot. Proc. Natl Acad. Sci. USA 87:3348.[Abstract/Free Full Text]
37. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl Acad. Sci. USA 76:4350.[Abstract/Free Full Text]
38. Ehlers, S., Mielke, M. E., Blankenstein, T. and Hahn, H. 1992. Kinetic analysis of cytokine gene expression in the livers of naive and immune mice infected with Listeria monocytogenes. The immediate early phase in innate resistance and acquired immunity. J. Immunol. 149:3016.[Abstract]
39. Morath, S., Stadelmaier, A., Geyer, A., Schmidt, R. R. and Hartung, T. 2002. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J. Exp. Med. 195:1635.[Abstract/Free Full Text]
40. Deininger, S., Stadelmaier, A., von Aulock, S., Morath, S., Schmidt, R. R. and Hartung, T. 2003. Definition of structural prerequisites for lipoteichoic acid-inducible cytokine induction by synthetic derivatives. J. Immunol. 170:4134.[Abstract/Free Full Text]
41. Fischer, W. 1990. Bacterial phosphoglycolipids and lipoteichoic acid. In Kates, M., ed., Glycolipids, Phosphoglycolipids and Sulfoglycolipids, p. 123, Plenum Press, New York.
42. Shibata, K., Hasebe, A., Into, T., Yamada, M. and Watanabe, T. 2000. The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts. J. Immunol. 165:6538.[Abstract/Free Full Text]
43. Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N. and Weis, J. J. 2000. Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine Toll-like receptor 2. J. Immunol. 165:618.[Abstract/Free Full Text]
44. Hirschfeld, M., Weis, J. J., Toshchakov, V. et al. 2001. Signaling by Toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect. Immun. 69:1477.[Abstract/Free Full Text]
45. Kirikae, T., Nitta, T., Kirikae, F., Suda, Y., Kusumoto, S., Qureshi, N. and Nakano, M. 1999. Lipopolysaccharides (LPS) of oral black-pigmented bacteria induce tumor necrosis factor production by LPS-refractory C3H/HeJ macrophages in a way different from that of Salmonella LPS. Infect. Immun. 67:1736.[Abstract/Free Full Text]
46. Asai, Y., Hashimoto, M., Fletcher, H. M., Miyake, K., Akira, S. and Ogawa, T. 2005. Lipopolysaccharide preparation extracted from Porphyromonas gingivalis lipoprotein-deficient mutant shows a marked decrease in Toll-like receptor 2-mediated signaling. Infect. Immun. 73:2157.[Abstract/Free Full Text]
47. Stoll, H., Dengjel, J., Nerz, C. and Gotz, F. 2005. Staphylococcus aureus deficient in lipidation of prelipoproteins is attenuated in growth and immune activation. Infect. Immun. 73:2411.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				J. Geurtsen, S. Chedammi, J. Mesters, M. Cot, N. N. Driessen, T. Sambou, R. Kakutani, R. Ummels, J. Maaskant, H. Takata, et al. Identification of Mycobacterial {alpha}-Glucan As a Novel Ligand for DC-SIGN: Involvement of Mycobacterial Capsular Polysaccharides in Host Immune Modulation J. Immunol., October 15, 2009; 183(8): 5221 - 5231. [Abstract] [Full Text] [PDF]

				M. Schmaler, N. J. Jann, F. Ferracin, L. Z. Landolt, L. Biswas, F. Gotz, and R. Landmann Lipoproteins in Staphylococcus aureus Mediate Inflammation by TLR2 and Iron-Dependent Growth In Vivo J. Immunol., June 1, 2009; 182(11): 7110 - 7118. [Abstract] [Full Text] [PDF]

				K. Tawaratsumida, M. Furuyashiki, M. Katsumoto, Y. Fujimoto, K. Fukase, Y. Suda, and M. Hashimoto Characterization of N-terminal Structure of TLR2-activating Lipoprotein in Staphylococcus aureus J. Biol. Chem., April 3, 2009; 284(14): 9147 - 9152. [Abstract] [Full Text] [PDF]

				H. J. Warshakoon, M. R. Burns, and S. A. David Structure-Activity Relationships of Antimicrobial and Lipoteichoic Acid-Sequestering Properties in Polyamine Sulfonamides Antimicrob. Agents Chemother., January 1, 2009; 53(1): 57 - 62. [Abstract] [Full Text] [PDF]

				L. Sweet, W. Zhang, H. Torres-Fewell, A. Serianni, W. Boggess, and J. Schorey Mycobacterium avium Glycopeptidolipids Require Specific Acetylation and Methylation Patterns for Signaling through Toll-like Receptor 2 J. Biol. Chem., November 28, 2008; 283(48): 33221 - 33231. [Abstract] [Full Text] [PDF]

				S. Machata, S. Tchatalbachev, W. Mohamed, L. Jansch, T. Hain, and T. Chakraborty Lipoproteins of Listeria monocytogenes Are Critical for Virulence and TLR2-Mediated Immune Activation J. Immunol., August 1, 2008; 181(3): 2028 - 2035. [Abstract] [Full Text] [PDF]

				P. Henneke, S. Dramsi, G. Mancuso, K. Chraibi, E. Pellegrini, C. Theilacker, J. Hubner, S. Santos-Sierra, G. Teti, D. T. Golenbock, et al. Lipoproteins Are Critical TLR2 Activating Toxins in Group B Streptococcal Sepsis J. Immunol., May 1, 2008; 180(9): 6149 - 6158. [Abstract] [Full Text] [PDF]

				S. Thakran, H. Li, C. L. Lavine, M. A. Miller, J. E. Bina, X. R. Bina, and F. Re Identification of Francisella tularensis Lipoproteins That Stimulate the Toll-like Receptor (TLR) 2/TLR1 Heterodimer J. Biol. Chem., February 15, 2008; 283(7): 3751 - 3760. [Abstract] [Full Text] [PDF]

				H. Li, M. M. Nooh, M. Kotb, and F. Re Commercial peptidoglycan preparations are contaminated with superantigen-like activity that stimulates IL-17 production J. Leukoc. Biol., February 1, 2008; 83(2): 409 - 418. [Abstract] [Full Text] [PDF]

				H. S. Seo, S. M. Michalek, and M. H. Nahm Lipoteichoic Acid Is Important in Innate Immune Responses to Gram-Positive Bacteria Infect. Immun., January 1, 2008; 76(1): 206 - 213. [Abstract] [Full Text] [PDF]

				C. Hermann Review: Variability of host pathogen interaction Innate Immunity, August 1, 2007; 13(4): 199 - 218. [Abstract] [PDF]

				M. Hashimoto, M. Furuyashiki, R. Kaseya, Y. Fukada, M. Akimaru, K. Aoyama, T. Okuno, T. Tamura, T. Kirikae, F. Kirikae, et al. Evidence of Immunostimulating Lipoprotein Existing in the Natural Lipoteichoic Acid Fraction Infect. Immun., April 1, 2007; 75(4): 1926 - 1932. [Abstract] [Full Text] [PDF]

				W. A. Derbigny, S.-C. Hong, M. S. Kerr, M. Temkit, and R. M. Johnson Chlamydia muridarum Infection Elicits a Beta Interferon Response in Murine Oviduct Epithelial Cells Dependent on Interferon Regulatory Factor 3 and TRIF Infect. Immun., March 1, 2007; 75(3): 1280 - 1290. [Abstract] [Full Text] [PDF]

				I. Bekeredjian-Ding, S. Inamura, T. Giese, H. Moll, S. Endres, A. Sing, U. Zahringer, and G. Hartmann Staphylococcus aureus Protein A Triggers T Cell-Independent B Cell Proliferation by Sensitizing B Cells for TLR2 Ligands J. Immunol., March 1, 2007; 178(5): 2803 - 2812. [Abstract] [Full Text] [PDF]

				Y. Sun, A. G. Hise, C. M. Kalsow, and E. Pearlman Staphylococcus aureus-Induced Corneal Inflammation Is Dependent on Toll-Like Receptor 2 and Myeloid Differentiation Factor 88 Infect. Immun., September 1, 2006; 74(9): 5325 - 5332. [Abstract] [Full Text] [PDF]

				M. Hashimoto, K. Tawaratsumida, H. Kariya, A. Kiyohara, Y. Suda, F. Krikae, T. Kirikae, and F. Gotz Not Lipoteichoic Acid but Lipoproteins Appear to Be the Dominant Immunobiologically Active Compounds in Staphylococcus aureus. J. Immunol., September 1, 2006; 177(5): 3162 - 3169. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 18/2/355    most recent dxh374v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (14) Request Permissions Google Scholar Articles by Hashimoto, M. Articles by Suda, Y. Search for Related Content PubMed PubMed Citation Articles by Hashimoto, M. Articles by Suda, Y. Social Bookmarking          What's this?

Online ISSN 1460-2377 - Print ISSN 0953-8178

Copyright © 2009 Japanese Society for Immunology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics