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Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety
Basic Research—Biology
Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety Jung Eun Baik, BS,* Kyoung-Soon Jang, PhD,† Seok-Seong Kang, PhD,* Cheol-Heui Yun, PhD,‡ Kangseok Lee, PhD,§ Byung-Gee Kim, PhD,† Kee-Yeon Kum, DDS, PhD,k and Seung Hyun Han, PhD* Abstract Introduction: Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor a in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. Methods: LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ionexchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide–treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma–induced protein (IP-10) and macrophage inflammatory protein-1a (MIP-1a) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). Results: The production of NO, IP-10, and MIP-1a was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide–pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyldiacylglycerol consisting of two acyl chains with chain lengths of C16 to C22 and with one or two unsaturated double bonds. However, those peaks were not observed
in the mass spectra of the calcium hydroxide–treated LTA. Furthermore, free fatty acids released from the calcium hydroxide–treated LTA were detected using TLC. Conclusion: We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA. (J Endod 2011;37:191–196)
Key Words Apical periodontitis, calcium hydroxide, Enterococcus faecalis, intracanal medicament, lipoteichoic acid, matrix–assisted laser desorption ionization time of flight
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nterococci are commensal microorganisms that can be frequently found in the mucosal tissues in the oral cavity, gastrointestinal tract, and genital tract in humans. Enterococci are of clinical importance because they are the third most common nosocomial pathogens (1) and the cause of refractory apical periodontitis (2). They are often resistant to disinfectants and antiseptics because they can persist under harsh conditions, such as high alkalinity, because of their efficient use of proton pumps (3). To date, 12 enterococci species have been identified, and approximately 90% of the Enterococcus clinical isolates are Enterococcus faecalis (4). E. faecalis expresses various virulence factors including lipoteichoic acid (LTA), peptidoglycan, aggregation substance, surface adhesins, sex pheromones, lytic enzymes such as gelatinase and hyaluronidase, and cytolysin (5). Of these virulence factors, LTA is considered one of the most important etiologic factors that are closely involved in pathogenicity based on the following aspects: (i) LTA is responsible for inflammatory responses and tissue damages (6); (ii) LTA purified from E. faecalis is able to induce proinflammatory cytokines and nitric oxide (NO) (7); (iii) E. faecalis LTA contributes to the bacterial adherence and biofilm formation that are essential for bacterial resistance to disinfectants, antibiotics, and host antimicrobial molecules (8); and (iv) opsonic antibodies to E. faecalis are predominantly generated against epitopes on the LTA (9). LTA is an amphiphilic molecule composed of a glycolipid anchor together with either polyglycerolphosphates or polyribitolphosphates (10). Most gram-positive bacteria including E. faecalis express polyglycerophosphate-type LTA, whereas
From the *Department of Oral Microbiology & Immunology, Dental Research Institute, and BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea; †Institute of Molecular Biology and Genetics, Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea; ‡Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea; §Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea; and kDepartment of Conservative Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea. Supported by grants from Korea Research Foundation funded by the Korean Government (KRF-2008-314-E00223), from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0029116), and from a Science Research Center grant to the Bone Metabolism Research Center (20100001746) funded by the Korean Ministry of Education, Science and Technology, Republic of Korea. Address requests for reprints to Dr Seung Hyun Han, Department of Oral Microbiology & Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.11.007
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Basic Research—Biology line treatment almost completely abolishes the inflammatory and immunostimulatory capacities of LTA (12). In contrast, LTA with more acyl chains elicits stronger immunostimulating activities (13). Thus, the lipid moiety of LTA seems to be a molecular target for the control of infectious diseases. Calcium hydroxide is widely used as an intracanal medicament to eliminate microorganisms in infected root canals because of its broad antimicrobial activity (14). Accumulating reports suggest that calcium hydroxide in aqueous solution releases hydroxide ion (OH), resulting in a high alkaline environment in which most bacteria cannot survive (15). Recently, we found that calcium hydroxide could attenuate the abilities of not only E. faecalis but also its LTA to stimulate murine macrophages. Indeed, LTA treated with calcium hydroxide could not stimulate the production of tumor necrosis factor a (TNF-a) or NO (16). However, the molecular mechanisms behind the inactivation of LTA caused by calcium hydroxide treatment have not been studied. The aim of the present study was to examine if calcium hydroxide deacylates LTA of E. faecalis resulting in the loss of immunostimulating activity with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and thin layer chromatography (TLC).
Materials and Methods Reagents and Chemicals Highly pure LTA was prepared from E. faecalis ATCC 29212 as previously described (7). Calcium hydroxide was purchased from DC Chemical Co. Ltd. (Seoul, Korea). Dulbecco’s modified Eagle’s medium and fetal bovine serum were obtained from Invitrogen (Grand Island, NY) and HyClone (Logan, UT), respectively. Proteomics-grade water was purchased from Bio-RAD (Hercules, CA). Chloroform, acetic acid, and methanol were purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Treatment of E. faecalis LTA with Calcium Hydroxide Previously, it has been shown that 25 mg/mL of calcium hydroxide or 0.2 N of sodium hydroxide completely abolished the ability of LTA to stimulate the expression of inflammatory mediators via macrophages (16, 17). Thus, LTA (450 or 900 mg) was incubated with calcium hydroxide (25 mg/mL), sodium hydroxide (0.2 N), or proteomicsgrade water as a control for 60 minutes followed by neutralization to pH 7 with 1 N of HCl. Then, the samples were lyophilized and stored at 80 C until use.
Figure 1. Calcium hydroxide abolishes the ability of E. faecalis LTA to stimulate the expression of inflammatory mediators. LTA from E. faecalis was treated with or without calcium hydroxide (25 mg/mL) at 37 C for 60 minutes. After pH neutralization, the calcium hydroxide–treated LTA was used for the stimulation of RAW 264.7 cells (1 106 cells/mL) for 24 hours. At the end of the incubation, culture media were collected and analyzed for the production of (A) NO, (B) IP-10, and (C) MIP-1a. Bars indicate mean values standard deviation, and asterisks are noted for data significantly (p < 0.01) different from the LTA-treated group. One of three similar results is shown. ND, not detected.
a few species such as Streptococcus pneumoniae produce polyribitolphosphate-type LTA (11). Studies on the structural and functional relationships suggest that glycolipid is mainly responsible for the immunostimulatory potential of LTA. In fact, delipidation of LTA by alka192
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Culture of RAW 264.7 Cells The mouse macrophage cell line RAW 264.7 (TIB-71) was purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37 C in a humidified incubator with 5% CO2. Measurement of Inflammatory Mediators RAW 264.7 cells at 1 106 cells/mL were stimulated with intact or calcium hydroxide–treated LTA for 24 hours. At the end of the incubation period, culture media were collected and used for the determination of NO and chemokines, interferon-gamma–induced protein (IP10), and macrophage inflammatory protein-1a (MIP-1a). For NO determination, the culture media were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine JOE — Volume 37, Number 2, February 2011
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Figure 2. Mass spectra obtained from the glycolipid anchor of E. faecalis LTA. In order to disintegrate polyglycerophosphates of E. faecalis LTA, 450 mg LTA was solubilized and boiled in 98% acetic acid at 100 C for 3 hours followed by lyophilization. Then, a solvent comprised of chloroform, methanol, and water (1:1:0.9, v/v/v) was added to the lyophilized products and underwent vigorous mixing. After phase separation, the organic phase containing the glycolipids of LTA was collected and subjected to MALDI-TOF mass spectrometry. One of three independent results is shown.
dihydrochloride, and 2% phosphoric acid) and incubated for 5 minutes at room temperature. Then, the optical density was measured at 540 nm with a VERSAmax microtiter-plate reader (Molecular Devices, Sunnyvale, CA) using NaNO2 as a standard. Levels of IP-10 and MIP-1a were determined using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocols.
Preparation of Glycolipid Anchor from E. faecalis LTA The lyophilized LTA samples were exposed to 98% (v/v) acetic acid (1 mL) to cleave the phosphodiester bond on LTA, followed by sonication for 30 minutes, boiling at 100 C for 3 hours, and lyophilization. Then, a solvent comprised of chloroform, methanol, and water (1:1:0.9, v/v/v) was added to the lyophilized products, and the chloroform phase containing the glycolipid anchors of LTA was obtained.
Structural Characterization of the Glycolipid Derived from LTA Glycolipids were characterized using MALDI-TOF mass spectrometry coupled with BioTools software (Bruker Daltonics, Bremen, Germany). Briefly, the chloroform phase was concentrated to 10 mL under vacuum and was dissolved in 50 mL of chloroform methanol (1:1, v/v). Then, 1 mL of the sample was mixed with 1 mL of 30 mg/ mL 2,5-dihydroxybenzoic acid (Sigma-Aldrich) in 70% acetonitrile. One microliter of the resulting aliquots was then deposited onto a metallic sample holder, the solvents were removed under vacuum, and MALDI-TOF mass spectrometry was performed using Biflex IV systems (Bruker Daltonics). JOE — Volume 37, Number 2, February 2011
TLC for Examination of Fatty Acids TLC was performed on a silica gel TLC plate (Silica gel 60; Merck, Whitehouse Station, NJ) with a solvent comprised of chloroform, methanol, and water at 60:30:5 (v/v/v). Glycolipids and released free fatty acid on the TLC plate were visualized by spraying with 5% phosphomolybdic acid in ethanol followed by heating at 120 C. Statistical Analysis All experiments were performed at least three times. The mean value standard deviation was determined from each treatment group in a given experiment. Statistical significance was examined by comparing the test group with a corresponding control group using a two-tailed t test. Differences were considered significant when p < 0.01.
Results Calcium Hydroxide Inhibits the Ability of E. faecalis LTA to Induce the Production of Inflammatory Mediators in Murine Macrophages Our previous study showed that E. faecalis LTA stimulates murine macrophages to release TNF-a, but pretreatment of LTA with calcium hydroxide did not show such an effect (16). To validate and generalize the hypothesis that calcium hydroxide is able to attenuate the inflammatory ability of E. faecalis LTA, RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide–treated LTA for 24 hours, and the culture media were analyzed for the productions of NO, IP-10, and MIP-1a. As shown in Fig. 1, the intact LTA augmented the expressions of inflammatory mediators, but pretreatment of LTA with calcium hydroxide abolished their expressions (Fig. 1). No significant change in cell viability was observed in any treatment group as determined Ca(OH)2 Inactivates LTA from E. faecalis
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Figure 3. Calcium hydroxide delipidates LTA of E. faecalis. LTA at 450 mg was incubated in 25 mg/mL calcium hydroxide or 0.2 N sodium hydroxide at 37 C for 60 minutes, and their glycolipids were analyzed using MALDI-TOF mass spectrometry. The panels present MALDI-TOF mass spectra for (A) proteomics-grade water, (B) the glycolipid fraction from LTA treated with proteomics-grade water, (C) calcium hydroxide at 25 mg/mL, (D) the glycolipid fraction from LTA treated with 25 mg/mL calcium hydroxide, (E) sodium hydroxide at 0.2 N, and (F) the glycolipid fraction from LTA treated with 0.2 N sodium hydroxide. One of three representative results is shown.
by MTT assay (data not shown). These results suggest that calcium hydroxide could attenuate the capacity of E. faecalis LTA to induce the production of proinflammatory mediators.
Structural Characterization of Glycolipid Anchors from E. faecalis LTA Accumulating reports suggest that glycolipids are pivotal for the immunostimulating potential of LTA (12, 17). Despite the importance of glycolipid, the molecular structure in relation to E. faecalis is still unclear. Therefore, we analyzed the glycolipid molecular structures of the LTA with or without calcium hydroxide treatment using MALDITOF mass spectrometry. Mass spectra showed that peaks were mainly observed from 930 to 1,070 mass units (m/z) (Fig. 2). Among the distinct peaks, 12 representative peaks were analyzed with BioTools software for molecular weight–based predictions of their structures. The glycolipids were shown to be dihexosyl diacylglycerol, in which the acyl chain lengths varied from C16 to C22. Interestingly, not only satu194
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rated and monounsaturated fatty acids but also diunsaturated fatty acids were observed.
Calcium Hydroxide Deacylates the Glycolipid Moiety of LTA from E. faecalis We hypothesized that calcium hydroxide can modify the LTA structure, in particular the glycolipid moiety, based on the idea that calcium hydroxide elicits high alkalinity through the release of OH- because sodium hydroxide could also delipidate the LTA structure (15, 18). Hence, we examined whether calcium hydroxide could deacylate the glycolipid anchor of LTA from E. faecalis using MALDI-TOF mass spectrometry. As shown in Fig. 3B, peaks for glycolipid anchors from E. faecalis LTA were detected on the mass spectra in an expected manner. However, such peaks were not observed in the glycolipid fractions of calcium hydroxide–treated (Fig. 3D) or sodium hydroxide– treated LTA (Fig. 3E). Notably, the glycolipids undergoing deacylation cannot be detected within the m/z ranges at 930 to 1,070 because they fragment into small pieces of free fatty acids, probably with JOE — Volume 37, Number 2, February 2011
Basic Research—Biology
Figure 4. Identifications of free fatty acids generated from the glycolipids after calcium hydroxide treatment. E. faecalis LTA (900 mg) was incubated in 25 mg/mL calcium hydroxide or 0.2 N sodium hydroxide at 37 C for 60 minutes, and their glycolipids were prepared as described in the Materials and Methods section. TLC was performed in a solvent containing chloroform, methanol, and water (60:30:5, v/v/v), and fatty acids were visualized with 5% ethanolic phosphomolybdic acid. Lane 1, glycolipid from E. faecalis LTA; Lane 2, glycolipid from calcium hydroxide–treated E. faecalis LTA; Lane 3, glycolipid from sodium hydroxide–treated E. faecalis LTA. The positions of the origin and TLC solvent front are indicated by arrows. One of three similar results is shown. Rf, retention factor.
a minimum size of 250.230 Da (for C16:2) to a maximum size of 338.355 Da (for C22) and a remaining dihexose with a glycerol backbone at 384.090 Da. In order to confirm the calcium hydroxide–mediated deacylation of LTA, we performed TLC analysis to examine the release of fatty acids from the glycolipids in the E. faecalis LTA treated with calcium hydroxide. As shown in Fig. 4, various glycolipids from the intact LTA were detected in ladders via TLC because of differences in the lengths and saturation degrees of the fatty acid chains. However, free fatty acids released from the calcium hydroxide–treated LTA were detected in identical patterns as those of free fatty acids from LTA deacylated by sodium hydroxide treatment (Fig. 4). Collectively, these results suggest that calcium hydroxide deacylates the LTA structure, leading to abolishment of the inflammatory activity of E. faecalis LTA.
Discussion Although we have previously shown that calcium hydroxide could attenuate the ability of E. faecalis LTA to induce proinflammatory cytokines in macrophages (16), the structural changes in LTA during calcium hydroxide treatment had not been investigated. In the present study, we found that calcium hydroxide could deacylate the LTA from E. faecalis, resulting in the impairment of LTA immunostimulating activity. The results provide an important insight for understanding the molecular mechanisms by which calcium hydroxide inactivates LTA of E. faecalis. Our results suggest that the glycolipid moiety plays a pivotal role in the immunostimulatory capacity of LTA. This hypothesis is further supJOE — Volume 37, Number 2, February 2011
ported by previous studies as followings. First, our recent study using xray crystallography showed that acyl chains were directly involved in the binding of LTA to its recognition receptor, Toll-like receptor 2 (19). Second, deacylated LTA could not stimulate Toll-like receptor 2 (20). Third, deacylated LTA could not induce the expression of proinflammatory mediators (12). Fourth, studies using synthetic LTA showed that glycolipid was critical for the immunostimulatory activity of LTA (12). Finally, Enterococcus hirae ATCC 9790 expressed two kinds of LTA, diacylated and tetraacylated, the latter of which is more potent than the former with regard to immunostimulatory potential (21). The structure of E. faecalis LTA has been relatively well characterized except for its composition of fatty acids. Its hydrophilic chains are repetitious 1,3-polyglycerolphosphates; the C-2 position of the glycerol residues are nonstoichiometrically substituted with hydroxy, D-Ala, kojibiose, or [D-Ala/6]-a-D-Glcp-(1/2-[D-Ala/6]-a-D-Glcp1/) (9). The structure of the glycolipid anchor is Glc-a-(1/2)Glc-a-(1/3)-diacylglycerol, and the fatty acids are saturated straight chains and cis-monounsaturated chains (22). The MALDI-TOF mass spectra of our study also suggest that the glycolipids from E. faecalis LTA consist of dihexosyl diacylglycerol harboring fatty acids ranging from C16 to C22. However, interestingly, the fatty acid structures are likely to be not only saturated and monounsaturated chains but also diunsaturated chains. One possible explanation for the detection of the diunsaturated LTA in our study is that LTA with diunsaturated fatty acids may have been in a negligible amount in their previous study (22) because most LTAs probably possessed saturated and monounsaturated acyl chains. Coincidently, our mass spectra also showed that the diunsaturated LTAs were detected at mass peaks with low intensity as shown in Fig. 2. Another possible explanation is the difference in E. faecalis strains that were used in our study (ATCC 29212) and their study (DSM 20371 and 20478) (22). Remarkably, E. faecalis can produce LTA with two or four fatty acids dependent on the strain (22). On the other hand, it should be further investigated if both acyl chains possess one of each double bond or if one is saturated and the other acyl chain has two double bonds. The structures of the fatty acids appear to be important in the determination of the immunostimulating potentials of LTAs. Accumulating results suggest that LTAs with unsaturated fatty acids tend to be less potent than LTAs with saturated fatty acids. Pneumococcal LTA comprised of unsaturated fatty acids is weaker than staphylococcal LTA comprised of all saturated fatty acids (20). LTA from Lactobacillus plantarum hardly induces the expression of inflammatory mediators in macrophages (17), and the structural analysis using mass spectrometry suggests that its glycolipid also contains unsaturated fatty acids (unpublished data). E. faecalis LTA was also less potent than staphylococcal LTA based on the fact that staphylococcal LTA alone sufficiently induced NO production (23), whereas LTA from E. faecalis required interferong for sufficient induction of NO production (7). The differential immunostimulating potentials are likely caused by differences in the affinities to lipid rafts, which are highly ordered phospholipid microdomains enriched with saturated phospholipid and cholesterol (24). The formation of lipid rafts is required for the stimulation of immune cells by various virulence factors, including lipopolysaccharide (LPS) (25, 26). Indeed, polyunsaturated fatty acids disturb immunostimulatory signals by altering the structures of lipid rafts (27, 28). Thus, LTA with unsaturated fatty acids might have a lower affinity for lipid rafts, possibly resulting in lower immunostimulatory potential. Calcium hydroxide can also inactivate LPS in gram-negative bacteria (29), which is considered as a counterpart of LTA because of structural and functional similarity. Indeed, calcium hydroxide– treated LPS failed to induce the production of TNF-a from monocytes (30) and the stimulation of osteoclast formation (31). Concomitant Ca(OH)2 Inactivates LTA from E. faecalis
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References 1. Wisplinghoff H, Seifert H, Tallent SM, et al. Nosocomial bloodstream infections in pediatric patients in United States hospitals: epidemiology, clinical features and susceptibilities. Pediatr Infect Dis J 2003;22:686–91. 2. Hancock HH 3rd, Sigurdsson A, Trope M, et al. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579–86. 3. Evans M, Davies JK, Sundqvist G, et al. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J 2002;35:221–8. 4. Gordon S, Swenson JM, Hill BC, et al. Antimicrobial susceptibility patterns of common and unusual species of enterococci causing infections in the United States. Enterococcal Study Group. J Clin Microbiol 1992;30:2373–8. 5. Kayaoglu G, Orstavik D. Virulence factors of Enterococcus faecalis: relationship to endodontic disease. Crit Rev Oral Biol Med 2004;15:308–20. 6. Ginsburg I. Role of lipoteichoic acid in infection and inflammation. Lancet Infect Dis 2002;2:171–9. 7. Baik JE, Ryu YH, Han JY, et al. Lipoteichoic acid partially contributes to the inflammatory responses to Enterococcus faecalis. J Endod 2008;34:975–82. 8. Fabretti F, Theilacker C, Baldassarri L, et al. Alanine esters of enterococcal lipoteichoic acid play a role in biofilm formation and resistance to antimicrobial peptides. Infect Immun 2006;74:4164–71. 9. Theilacker C, Kaczynski Z, Kropec A, et al. Opsonic antibodies to Enterococcus faecalis strain 12030 are directed against lipoteichoic acid. Infect Immun 2006;74: 5703–12. 10. Wicken AJ, Knox KW. Lipoteichoic acids: a new class of bacterial antigen. Science 1975;187:1161–7. 11. Seo HS, Cartee RT, Pritchard DG, et al. A new model of pneumococcal lipoteichoic acid structure resolves biochemical, biosynthetic, and serologic inconsistencies of the current model. J Bacteriol 2008;190:2379–87. 12. Morath S, Stadelmaier A, Geyer A, et al. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release. J Exp Med 2002;195: 1635–40. 13. Hashimoto M, Imamura Y, Yasuoka J, et al. A novel cytokine-inducing glycolipid isolated from the lipoteichoic acid fraction of Enterococcus hirae ATCC 9790: a fundamental structure of the hydrophilic part. Glycoconj J 1999;16:213–21. 14. Stuart KG, Miller CH, Brown CE Jr, et al. The comparative antimicrobial effect of calcium hydroxide. Oral Surg Oral Med Oral Pathol 1991;72:101–4. 15. Gomes BP, Ferraz CC, Vianna ME, et al. In vitro antimicrobial activity of calcium hydroxide pastes and their vehicles against selected microorganisms. Braz Dent J 2002;13:155–61.
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16. Baik JE, Kum KY, Yun CH, et al. Calcium hydroxide inactivates lipoteichoic acid from Enterococcus faecalis. J Endod 2008;34:1355–9. 17. Ryu YH, Baik JE, Yang JS, et al. Differential immunostimulatory effects of grampositive bacteria due to their lipoteichoic acids. Int Immunopharmacol 2009;9: 127–33. 18. Kim JH, Seo H, Han SH, et al. Monoacyl lipoteichoic acid from pneumococci stimulates human cells but not mouse cells. Infect Immun 2005;73:834–40. 19. Kang JY, Nan X, Jin MS, et al. Recognition of lipopeptide patterns by Toll-like receptor 2-Toll-like receptor 6 heterodimer. Immunity 2009;31:873–84. 20. Han SH, Kim JH, Martin M, et al. Pneumococcal lipoteichoic acid (LTA) is not as potent as staphylococcal LTA in stimulating Toll-like receptor 2. Infect Immun 2003;71:5541–8. 21. Takada H, Kawabata Y, Arakaki R, et al. Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine-inducing, antitumor, and antigenic activities. Infect Immun 1995;63:57–65. 22. Hermann C, Spreitzer I, Schroder NW, et al. Cytokine induction by purified lipoteichoic acids from various bacterial species—role of LBP, sCD14, CD14 and failure to induce IL-12 and subsequent IFN-gamma release. Eur J Immunol 2002;32:541–51. 23. Han SH, Kim JH, Seo HS, et al. Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2. J Immunol 2006;176:573–9. 24. Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest 2002;110: 597–603. 25. Fessler MB, Arndt PG, Frasch SC, et al. Lipid rafts regulate lipopolysaccharideinduced activation of Cdc42 and inflammatory functions of the human neutrophil. J Biol Chem 2004;279:39989–98. 26. Triantafilou M, Miyake K, Golenbock DT, et al. Mediators of innate immune recognition of bacteria concentrate in lipid rafts and facilitate lipopolysaccharide-induced cell activation. J Cell Sci 2002;115:2603–11. 27. Stulnig TM, Berger M, Sigmund T, et al. Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains. J Cell Biol 1998;143:637–44. 28. Stulnig TM, Huber J, Leitinger N, et al. Polyunsaturated eicosapentaenoic acid displaces proteins from membrane rafts by altering raft lipid composition. J Biol Chem 2001;276:37335–40. 29. Safavi KE, Nichols FC. Effect of calcium hydroxide on bacterial lipopolysaccharide. J Endod 1993;19:76–8. 30. Barthel CR, Levin LG, Reisner HM, et al. TNF-alpha release in monocytes after exposure to calcium hydroxide treated Escherichia coli LPS. Int Endod J 1997;30: 155–9. 31. Jiang J, Zuo J, Chen SH, et al. Calcium hydroxide reduces lipopolysaccharidestimulated osteoclast formation. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;95:348–54. 32. Buck RA, Cai J, Eleazer PD, et al. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2001;27:325–7. 33. Lee JK, Baik JE, Yun CH, et al. Chlorhexidine gluconate attenuates the ability of lipoteichoic acid from Enterococcus faecalis to stimulate toll-like receptor 2. J Endod 2009;35:212–5. 34. Zorko M, Jerala R. Alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid and prevent cell activation by antibiotics. J Antimicrob Chemother 2008;62:730–7.
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Calcium Hydroxide Inactivates Lipoteichoic Acid from Enterococcus faecalis through Deacylation of the Lipid Moiety Jung Eun Baik, BS,* Kyoung-Soon Jang, PhD,† Seok-Seong Kang, PhD,* Cheol-Heui Yun, PhD,‡ Kangseok Lee, PhD,§ Byung-Gee Kim, PhD,† Kee-Yeon Kum, DDS, PhD,k and Seung Hyun Han, PhD* Abstract Introduction: Lipoteichoic acid (LTA) is a major virulence factor of Enterococcus faecalis that is closely associated with refractory apical periodontitis. Recently, we have shown that calcium hydroxide, a commonly used intracanal medicament, abrogated the ability of LTA to stimulate the production of tumor necrosis factor a in a murine macrophage line, RAW 264.7. Because calcium hydroxide could potentially modify the glycolipid moiety of LTA, we examined if calcium hydroxide inactivates LTA through deacylation of the LTA. Methods: LTA was prepared from E. faecalis by organic solvent extraction followed by chromatography with the hydrophobic-interaction column and the ionexchange column. RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide–treated LTA for 24 hours, and the productions of nitric oxide (NO) and chemokines interferon-gamma–induced protein (IP-10) and macrophage inflammatory protein-1a (MIP-1a) were determined. The glycolipid structure of LTA was analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry and thin layer chromatography (TLC). Results: The production of NO, IP-10, and MIP-1a was augmented in LTA-stimulated cells, whereas no such effect was observed upon stimulation with calcium hydroxide–pretreated LTA. Mass spectrometry showed that intact glycolipids of LTA yielded distinct mass peaks at 930 to 1,070 mass over charge (m/z) units, corresponding to dihexosyldiacylglycerol consisting of two acyl chains with chain lengths of C16 to C22 and with one or two unsaturated double bonds. However, those peaks were not observed
in the mass spectra of the calcium hydroxide–treated LTA. Furthermore, free fatty acids released from the calcium hydroxide–treated LTA were detected using TLC. Conclusion: We suggest that calcium hydroxide attenuates the inflammatory activity of E. faecalis LTA through deacylation of the LTA. (J Endod 2011;37:191–196)
Key Words Apical periodontitis, calcium hydroxide, Enterococcus faecalis, intracanal medicament, lipoteichoic acid, matrix–assisted laser desorption ionization time of flight
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nterococci are commensal microorganisms that can be frequently found in the mucosal tissues in the oral cavity, gastrointestinal tract, and genital tract in humans. Enterococci are of clinical importance because they are the third most common nosocomial pathogens (1) and the cause of refractory apical periodontitis (2). They are often resistant to disinfectants and antiseptics because they can persist under harsh conditions, such as high alkalinity, because of their efficient use of proton pumps (3). To date, 12 enterococci species have been identified, and approximately 90% of the Enterococcus clinical isolates are Enterococcus faecalis (4). E. faecalis expresses various virulence factors including lipoteichoic acid (LTA), peptidoglycan, aggregation substance, surface adhesins, sex pheromones, lytic enzymes such as gelatinase and hyaluronidase, and cytolysin (5). Of these virulence factors, LTA is considered one of the most important etiologic factors that are closely involved in pathogenicity based on the following aspects: (i) LTA is responsible for inflammatory responses and tissue damages (6); (ii) LTA purified from E. faecalis is able to induce proinflammatory cytokines and nitric oxide (NO) (7); (iii) E. faecalis LTA contributes to the bacterial adherence and biofilm formation that are essential for bacterial resistance to disinfectants, antibiotics, and host antimicrobial molecules (8); and (iv) opsonic antibodies to E. faecalis are predominantly generated against epitopes on the LTA (9). LTA is an amphiphilic molecule composed of a glycolipid anchor together with either polyglycerolphosphates or polyribitolphosphates (10). Most gram-positive bacteria including E. faecalis express polyglycerophosphate-type LTA, whereas
From the *Department of Oral Microbiology & Immunology, Dental Research Institute, and BK21 Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea; †Institute of Molecular Biology and Genetics, Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Republic of Korea; ‡Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea; §Department of Life Science, Chung-Ang University, Seoul 156-756, Republic of Korea; and kDepartment of Conservative Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea. Supported by grants from Korea Research Foundation funded by the Korean Government (KRF-2008-314-E00223), from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0029116), and from a Science Research Center grant to the Bone Metabolism Research Center (20100001746) funded by the Korean Ministry of Education, Science and Technology, Republic of Korea. Address requests for reprints to Dr Seung Hyun Han, Department of Oral Microbiology & Immunology, Dental Research Institute and BK21 Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Republic of Korea. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2011 American Association of Endodontists. doi:10.1016/j.joen.2010.11.007
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Materials and Methods Reagents and Chemicals Highly pure LTA was prepared from E. faecalis ATCC 29212 as previously described (7). Calcium hydroxide was purchased from DC Chemical Co. Ltd. (Seoul, Korea). Dulbecco’s modified Eagle’s medium and fetal bovine serum were obtained from Invitrogen (Grand Island, NY) and HyClone (Logan, UT), respectively. Proteomics-grade water was purchased from Bio-RAD (Hercules, CA). Chloroform, acetic acid, and methanol were purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Treatment of E. faecalis LTA with Calcium Hydroxide Previously, it has been shown that 25 mg/mL of calcium hydroxide or 0.2 N of sodium hydroxide completely abolished the ability of LTA to stimulate the expression of inflammatory mediators via macrophages (16, 17). Thus, LTA (450 or 900 mg) was incubated with calcium hydroxide (25 mg/mL), sodium hydroxide (0.2 N), or proteomicsgrade water as a control for 60 minutes followed by neutralization to pH 7 with 1 N of HCl. Then, the samples were lyophilized and stored at 80 C until use.
Figure 1. Calcium hydroxide abolishes the ability of E. faecalis LTA to stimulate the expression of inflammatory mediators. LTA from E. faecalis was treated with or without calcium hydroxide (25 mg/mL) at 37 C for 60 minutes. After pH neutralization, the calcium hydroxide–treated LTA was used for the stimulation of RAW 264.7 cells (1 106 cells/mL) for 24 hours. At the end of the incubation, culture media were collected and analyzed for the production of (A) NO, (B) IP-10, and (C) MIP-1a. Bars indicate mean values standard deviation, and asterisks are noted for data significantly (p < 0.01) different from the LTA-treated group. One of three similar results is shown. ND, not detected.
a few species such as Streptococcus pneumoniae produce polyribitolphosphate-type LTA (11). Studies on the structural and functional relationships suggest that glycolipid is mainly responsible for the immunostimulatory potential of LTA. In fact, delipidation of LTA by alka192
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Culture of RAW 264.7 Cells The mouse macrophage cell line RAW 264.7 (TIB-71) was purchased from the American Type Culture Collection (Manassas, VA). The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 U/mL of penicillin, and 100 mg/mL of streptomycin at 37 C in a humidified incubator with 5% CO2. Measurement of Inflammatory Mediators RAW 264.7 cells at 1 106 cells/mL were stimulated with intact or calcium hydroxide–treated LTA for 24 hours. At the end of the incubation period, culture media were collected and used for the determination of NO and chemokines, interferon-gamma–induced protein (IP10), and macrophage inflammatory protein-1a (MIP-1a). For NO determination, the culture media were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine JOE — Volume 37, Number 2, February 2011
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Figure 2. Mass spectra obtained from the glycolipid anchor of E. faecalis LTA. In order to disintegrate polyglycerophosphates of E. faecalis LTA, 450 mg LTA was solubilized and boiled in 98% acetic acid at 100 C for 3 hours followed by lyophilization. Then, a solvent comprised of chloroform, methanol, and water (1:1:0.9, v/v/v) was added to the lyophilized products and underwent vigorous mixing. After phase separation, the organic phase containing the glycolipids of LTA was collected and subjected to MALDI-TOF mass spectrometry. One of three independent results is shown.
dihydrochloride, and 2% phosphoric acid) and incubated for 5 minutes at room temperature. Then, the optical density was measured at 540 nm with a VERSAmax microtiter-plate reader (Molecular Devices, Sunnyvale, CA) using NaNO2 as a standard. Levels of IP-10 and MIP-1a were determined using commercial enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocols.
Preparation of Glycolipid Anchor from E. faecalis LTA The lyophilized LTA samples were exposed to 98% (v/v) acetic acid (1 mL) to cleave the phosphodiester bond on LTA, followed by sonication for 30 minutes, boiling at 100 C for 3 hours, and lyophilization. Then, a solvent comprised of chloroform, methanol, and water (1:1:0.9, v/v/v) was added to the lyophilized products, and the chloroform phase containing the glycolipid anchors of LTA was obtained.
Structural Characterization of the Glycolipid Derived from LTA Glycolipids were characterized using MALDI-TOF mass spectrometry coupled with BioTools software (Bruker Daltonics, Bremen, Germany). Briefly, the chloroform phase was concentrated to 10 mL under vacuum and was dissolved in 50 mL of chloroform methanol (1:1, v/v). Then, 1 mL of the sample was mixed with 1 mL of 30 mg/ mL 2,5-dihydroxybenzoic acid (Sigma-Aldrich) in 70% acetonitrile. One microliter of the resulting aliquots was then deposited onto a metallic sample holder, the solvents were removed under vacuum, and MALDI-TOF mass spectrometry was performed using Biflex IV systems (Bruker Daltonics). JOE — Volume 37, Number 2, February 2011
TLC for Examination of Fatty Acids TLC was performed on a silica gel TLC plate (Silica gel 60; Merck, Whitehouse Station, NJ) with a solvent comprised of chloroform, methanol, and water at 60:30:5 (v/v/v). Glycolipids and released free fatty acid on the TLC plate were visualized by spraying with 5% phosphomolybdic acid in ethanol followed by heating at 120 C. Statistical Analysis All experiments were performed at least three times. The mean value standard deviation was determined from each treatment group in a given experiment. Statistical significance was examined by comparing the test group with a corresponding control group using a two-tailed t test. Differences were considered significant when p < 0.01.
Results Calcium Hydroxide Inhibits the Ability of E. faecalis LTA to Induce the Production of Inflammatory Mediators in Murine Macrophages Our previous study showed that E. faecalis LTA stimulates murine macrophages to release TNF-a, but pretreatment of LTA with calcium hydroxide did not show such an effect (16). To validate and generalize the hypothesis that calcium hydroxide is able to attenuate the inflammatory ability of E. faecalis LTA, RAW 264.7 cells were stimulated with intact LTA or calcium hydroxide–treated LTA for 24 hours, and the culture media were analyzed for the productions of NO, IP-10, and MIP-1a. As shown in Fig. 1, the intact LTA augmented the expressions of inflammatory mediators, but pretreatment of LTA with calcium hydroxide abolished their expressions (Fig. 1). No significant change in cell viability was observed in any treatment group as determined Ca(OH)2 Inactivates LTA from E. faecalis
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Figure 3. Calcium hydroxide delipidates LTA of E. faecalis. LTA at 450 mg was incubated in 25 mg/mL calcium hydroxide or 0.2 N sodium hydroxide at 37 C for 60 minutes, and their glycolipids were analyzed using MALDI-TOF mass spectrometry. The panels present MALDI-TOF mass spectra for (A) proteomics-grade water, (B) the glycolipid fraction from LTA treated with proteomics-grade water, (C) calcium hydroxide at 25 mg/mL, (D) the glycolipid fraction from LTA treated with 25 mg/mL calcium hydroxide, (E) sodium hydroxide at 0.2 N, and (F) the glycolipid fraction from LTA treated with 0.2 N sodium hydroxide. One of three representative results is shown.
by MTT assay (data not shown). These results suggest that calcium hydroxide could attenuate the capacity of E. faecalis LTA to induce the production of proinflammatory mediators.
Structural Characterization of Glycolipid Anchors from E. faecalis LTA Accumulating reports suggest that glycolipids are pivotal for the immunostimulating potential of LTA (12, 17). Despite the importance of glycolipid, the molecular structure in relation to E. faecalis is still unclear. Therefore, we analyzed the glycolipid molecular structures of the LTA with or without calcium hydroxide treatment using MALDITOF mass spectrometry. Mass spectra showed that peaks were mainly observed from 930 to 1,070 mass units (m/z) (Fig. 2). Among the distinct peaks, 12 representative peaks were analyzed with BioTools software for molecular weight–based predictions of their structures. The glycolipids were shown to be dihexosyl diacylglycerol, in which the acyl chain lengths varied from C16 to C22. Interestingly, not only satu194
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rated and monounsaturated fatty acids but also diunsaturated fatty acids were observed.
Calcium Hydroxide Deacylates the Glycolipid Moiety of LTA from E. faecalis We hypothesized that calcium hydroxide can modify the LTA structure, in particular the glycolipid moiety, based on the idea that calcium hydroxide elicits high alkalinity through the release of OH- because sodium hydroxide could also delipidate the LTA structure (15, 18). Hence, we examined whether calcium hydroxide could deacylate the glycolipid anchor of LTA from E. faecalis using MALDI-TOF mass spectrometry. As shown in Fig. 3B, peaks for glycolipid anchors from E. faecalis LTA were detected on the mass spectra in an expected manner. However, such peaks were not observed in the glycolipid fractions of calcium hydroxide–treated (Fig. 3D) or sodium hydroxide– treated LTA (Fig. 3E). Notably, the glycolipids undergoing deacylation cannot be detected within the m/z ranges at 930 to 1,070 because they fragment into small pieces of free fatty acids, probably with JOE — Volume 37, Number 2, February 2011
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Figure 4. Identifications of free fatty acids generated from the glycolipids after calcium hydroxide treatment. E. faecalis LTA (900 mg) was incubated in 25 mg/mL calcium hydroxide or 0.2 N sodium hydroxide at 37 C for 60 minutes, and their glycolipids were prepared as described in the Materials and Methods section. TLC was performed in a solvent containing chloroform, methanol, and water (60:30:5, v/v/v), and fatty acids were visualized with 5% ethanolic phosphomolybdic acid. Lane 1, glycolipid from E. faecalis LTA; Lane 2, glycolipid from calcium hydroxide–treated E. faecalis LTA; Lane 3, glycolipid from sodium hydroxide–treated E. faecalis LTA. The positions of the origin and TLC solvent front are indicated by arrows. One of three similar results is shown. Rf, retention factor.
a minimum size of 250.230 Da (for C16:2) to a maximum size of 338.355 Da (for C22) and a remaining dihexose with a glycerol backbone at 384.090 Da. In order to confirm the calcium hydroxide–mediated deacylation of LTA, we performed TLC analysis to examine the release of fatty acids from the glycolipids in the E. faecalis LTA treated with calcium hydroxide. As shown in Fig. 4, various glycolipids from the intact LTA were detected in ladders via TLC because of differences in the lengths and saturation degrees of the fatty acid chains. However, free fatty acids released from the calcium hydroxide–treated LTA were detected in identical patterns as those of free fatty acids from LTA deacylated by sodium hydroxide treatment (Fig. 4). Collectively, these results suggest that calcium hydroxide deacylates the LTA structure, leading to abolishment of the inflammatory activity of E. faecalis LTA.
Discussion Although we have previously shown that calcium hydroxide could attenuate the ability of E. faecalis LTA to induce proinflammatory cytokines in macrophages (16), the structural changes in LTA during calcium hydroxide treatment had not been investigated. In the present study, we found that calcium hydroxide could deacylate the LTA from E. faecalis, resulting in the impairment of LTA immunostimulating activity. The results provide an important insight for understanding the molecular mechanisms by which calcium hydroxide inactivates LTA of E. faecalis. Our results suggest that the glycolipid moiety plays a pivotal role in the immunostimulatory capacity of LTA. This hypothesis is further supJOE — Volume 37, Number 2, February 2011
ported by previous studies as followings. First, our recent study using xray crystallography showed that acyl chains were directly involved in the binding of LTA to its recognition receptor, Toll-like receptor 2 (19). Second, deacylated LTA could not stimulate Toll-like receptor 2 (20). Third, deacylated LTA could not induce the expression of proinflammatory mediators (12). Fourth, studies using synthetic LTA showed that glycolipid was critical for the immunostimulatory activity of LTA (12). Finally, Enterococcus hirae ATCC 9790 expressed two kinds of LTA, diacylated and tetraacylated, the latter of which is more potent than the former with regard to immunostimulatory potential (21). The structure of E. faecalis LTA has been relatively well characterized except for its composition of fatty acids. Its hydrophilic chains are repetitious 1,3-polyglycerolphosphates; the C-2 position of the glycerol residues are nonstoichiometrically substituted with hydroxy, D-Ala, kojibiose, or [D-Ala/6]-a-D-Glcp-(1/2-[D-Ala/6]-a-D-Glcp1/) (9). The structure of the glycolipid anchor is Glc-a-(1/2)Glc-a-(1/3)-diacylglycerol, and the fatty acids are saturated straight chains and cis-monounsaturated chains (22). The MALDI-TOF mass spectra of our study also suggest that the glycolipids from E. faecalis LTA consist of dihexosyl diacylglycerol harboring fatty acids ranging from C16 to C22. However, interestingly, the fatty acid structures are likely to be not only saturated and monounsaturated chains but also diunsaturated chains. One possible explanation for the detection of the diunsaturated LTA in our study is that LTA with diunsaturated fatty acids may have been in a negligible amount in their previous study (22) because most LTAs probably possessed saturated and monounsaturated acyl chains. Coincidently, our mass spectra also showed that the diunsaturated LTAs were detected at mass peaks with low intensity as shown in Fig. 2. Another possible explanation is the difference in E. faecalis strains that were used in our study (ATCC 29212) and their study (DSM 20371 and 20478) (22). Remarkably, E. faecalis can produce LTA with two or four fatty acids dependent on the strain (22). On the other hand, it should be further investigated if both acyl chains possess one of each double bond or if one is saturated and the other acyl chain has two double bonds. The structures of the fatty acids appear to be important in the determination of the immunostimulating potentials of LTAs. Accumulating results suggest that LTAs with unsaturated fatty acids tend to be less potent than LTAs with saturated fatty acids. Pneumococcal LTA comprised of unsaturated fatty acids is weaker than staphylococcal LTA comprised of all saturated fatty acids (20). LTA from Lactobacillus plantarum hardly induces the expression of inflammatory mediators in macrophages (17), and the structural analysis using mass spectrometry suggests that its glycolipid also contains unsaturated fatty acids (unpublished data). E. faecalis LTA was also less potent than staphylococcal LTA based on the fact that staphylococcal LTA alone sufficiently induced NO production (23), whereas LTA from E. faecalis required interferong for sufficient induction of NO production (7). The differential immunostimulating potentials are likely caused by differences in the affinities to lipid rafts, which are highly ordered phospholipid microdomains enriched with saturated phospholipid and cholesterol (24). The formation of lipid rafts is required for the stimulation of immune cells by various virulence factors, including lipopolysaccharide (LPS) (25, 26). Indeed, polyunsaturated fatty acids disturb immunostimulatory signals by altering the structures of lipid rafts (27, 28). Thus, LTA with unsaturated fatty acids might have a lower affinity for lipid rafts, possibly resulting in lower immunostimulatory potential. Calcium hydroxide can also inactivate LPS in gram-negative bacteria (29), which is considered as a counterpart of LTA because of structural and functional similarity. Indeed, calcium hydroxide– treated LPS failed to induce the production of TNF-a from monocytes (30) and the stimulation of osteoclast formation (31). Concomitant Ca(OH)2 Inactivates LTA from E. faecalis
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