Human salivary proteins with affinity to lipoteichoic acid of Enterococcus faecalis

Molecular Immunology 77 (2016) 52–59

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Human salivary proteins with affinity to lipoteichoic acid of Enterococcus faecalis Jung Eun Baik a , Hyuk-Il Choe a , Sun Woong Hong a , Seok-Seong Kang a , Ki Bum Ahn a , Kun Cho b , Cheol-Heui Yun c , Seung Hyun Han a,∗ a Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul 08826, Republic of Korea b Biomedical Omics Group, Korea Basic Science Institute, Ochang 28119, Republic of Korea c Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea

a r t i c l e

i n f o

Article history: Received 30 April 2016 Received in revised form 15 July 2016 Accepted 19 July 2016 Keywords: Enterococcus faecalis Lipoteichoic acid Saliva Innate immunity

a b s t r a c t Enterococcus faecalis is associated with refractory apical periodontitis and its lipoteichoic acid (Ef.LTA) is considered as a major virulence factor. Although the binding proteins of Ef.LTA may play an important role for mediating infection and immunity in the oral cavity, little is known about Ef.LTA-binding proteins (Ef.LTA-BPs) in saliva. In this study, we identified salivary Ef.LTA-BPs with biotinylated Ef.LTA (Ef.LTAbiotin) through mass spectrometry. The biotinylation of Ef.LTA was confirmed by binding capacity with streptavidin-FITC on CHO/CD14/TLR2 cells. The biological activity of Ef.LTA-biotin was determined based on the induction of nitric oxide and macrophage inflammatory protein-1␣ in a macrophage cell-line, RAW 264.7. To identify salivary Ef.LTA-BPs, the Ef.LTA-biotin was mixed with a pool of human saliva obtained from nine healthy subjects followed by precipitation with a streptavidin-coated bead. Ef.LTA-BPs were then separated with 12% SDS-PAGE and subjected to the mass spectrometry. Six human salivary Ef.LTABPs including short palate lung and nasal epithelium carcinoma-associated protein 2, zymogen granule protein 16 homolog B, hemoglobin subunit ␣ and ␤, apolipoprotein A-I, and lipocalin-1 were identified with statistical significance (P < 0.05). Ef.LTA-BPs were validated with lipocalin-1 using pull-down assay. Hemoglobin inhibited the biofilm formation of E. faecalis whereas lipocalin-1 did not show such effect. Collectively, the identified Ef.LTA-BPs could provide clues for our understanding of the pathogenesis of E. faecalis and host immunity in oral cavity. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Enterococcus faecalis is a Gram-positive bacterium that inhabits the oral cavity, gastrointestinal tract, and genital tract of humans and animals (Jett et al., 1994). It is capable to survive in harsh environmental conditions such as high salt condition (Gardin et al., 2001), high alkalinity (Flahaut et al., 1997), and a diverse range of temperatures (Van Den Berghe et al., 2006). In the last decades, this bacterium has become one of the most common nosocomial pathogen (Wisplinghoff et al., 2003) because of its ability to resist against antimicrobial agents (Hunt, 1998) and its capacity to form biofilm on medical devices (Donlan and Costerton, 2002).

∗ Corresponding author at: Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Building 86, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea. E-mail address: [email protected] (S.H. Han). http://dx.doi.org/10.1016/j.molimm.2016.07.013 0161-5890/© 2016 Elsevier Ltd. All rights reserved.

On the other hand, in dentistry, it is considered a problematic pathogen due to its frequent recovery in teeth with refractory apical periodontitis (Hancock et al., 2001). E. faecalis expresses various virulence factors including lipoteichoic acid (LTA), peptidoglycan, and lytic enzymes such as gelatinase (Kayaoglu and Orstavik, 2004; Pynnonen et al., 2011). Among them, LTA plays a crucial role in bacterial adhesion, biofilm formation, and the induction of inflammatory mediators leading to tissue damage (Fabretti et al., 2006; Ginsburg, 2002). During Gram-positive bacterial infection, LTA is often recognized by the host LTA-binding proteins (LTA-BPs), which either facilitate LTA-mediated immune responses or neutralize the LTA. For example, CD14, L-ficolin and lipopolysaccharide-binding protein (LBP) are known as LTA-BPs involved in Toll-like receptor 2 (TLR2) signaling (Schroder et al., 2003) and complement activation (Lynch et al., 2004) for the induction of immune responses. Another LTA-BP, mannose-binding protein, removes LTA from the blood

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stream through its C-terminal carbohydrate recognition domain (Polotsky et al., 1996), and hemoglobin potentiates LTA-induced IL-6 production in murine macrophages (Hasty et al., 2006). Previously, we also identified several staphylococcal LTA-BPs in human serum using LTQ-Orbitrap hybrid Fourier transform mass spectrometry including neutrophil-activating peptide 2, prohibitin-2, alpha-1-anti-trypsin, histidine-rich glycoprotein, apolipoproteins, complement, and coagulation factors (Jang et al., 2012). Saliva plays an important role in the protection and maintenance of oral cavity by being involved in food digestion, lubrication, regulation of pH, and antimicrobial functions (Bennick et al., 1983; Lenander-Lumikari and Loimaranta, 2000). On the other hand, oral bacteria often use salivary proteins for their adhesion to tooth surfaces and invasion to the host (Scannapieco, 1994). Since the existence of E. faecalis in saliva is associated with the prevalence of E. faecalis in root canal, it is therefore crucial to identify salivary proteins that interact with E. faecalis LTA (Ef.LTA) to gain an understanding of the pathogenesis of E. faecalis and host immunity in the oral cavity. Although recently the human salivary proteins with affinity to Streptococcus mutans LTA have been reported including hemoglobulins, prolactin-inducible protein, histone H4, protein S100-A9, profilin-1, neutrophil defensing-1, and short palate lung and nasal epithelium carcinoma-associated protein 2 (SPLUNC2) (Hong et al., 2014), the Ef.LTA-binding proteins (Ef.LTA-BPs) in human saliva are yet to be identified. Thus, the purpose of this study was to identify salivary proteins with high affinity for Ef.LTA. 2. Materials and methods 2.1. Reagents and chemicals Highly-pure LTA was prepared from E. faecalis ATCC 29212 as previously described (Baik et al., 2008). Recombinant mouse IFN␥ and recombinant human lipocalin-1 were obtained from R&D Systems (Minneapolis, MS, USA). Neutravidin® agarose bead was purchased from Thermo Fisher scientific Inc. (Rockford, IL, USA). Biotin-3-sulfo-N-hydroxysuccinimide ester sodium salt (BiotinNHS) and all other reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. 2.2. Biotinylation of Ef.LTA with biotin-NHS

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2.5. Preparation of saliva samples The preparation of human saliva was approved by the Institutional Review Board of Seoul National University Dental Hospital (IRB No.CRI11008). The saliva samples from nine healthy subjects were prepared as described previously (Choi et al., 2011). 2.6. Identification of Ef.LTA-binding proteins (Ef.LTA-BPs) from human saliva After pre-incubating the pool of saliva obtained from nine healthy subjects with Neutravidin® agarose beads to remove nonspecific binding, the pre-cleared saliva (200 ␮g) was incubated with Ef.LTA (50 ␮g) or Ef.LTA-biotin (50 ␮g) with gentle agitation at 37 ◦ C for 1 h. Then, salivary Ef.LTA-BPs were isolated by incubating with Neutravidin® agarose beads (50 mg) at 4 ◦ C for 3 h. After spinning down and washing the beads with phosphate-buffered saline (PBS), the proteins bound to the agarose beads were eluted by incubating with 4 × sample buffer (8% SDS/10% 2-mercaptoethanol/30% glycerol/0.02% bromophenol blue in 0.25 M Tris-HCl, pH 6.8) at 100 ◦ C for 15 min. Then, the eluents were separated using SDSPAGE and visualized by silver staining as described previously (Choi et al., 2011). All bands in Ef.LTA-BPs were subjected to the mass spectrometric analysis at Korea Basic Science Institute (KBSI). The obtained MS and MS/MS spectra were analyzed with Mascot Daemon (Matrix Science, London, UK) using the IPI human database (IPI.HUMAN.v.3.73). Peptides were identified with a peptide tolerance of ± 50 ppm, fragment mass tolerance of ± 0.8 Da, and the consideration of two missed trypsin cleavage, oxidation of methionine, and fixed modification of carbamidomethyl cysteine. The isolation of Ef.LTA-BPs and mass spectrometric analysis was independently performed twice under the same condition. Peptide score is −10 × Log (P), where P indicates the probability that the observed match is a random event. Among the proteins comprising more than two peptides with over 36 peptide scores (P < 0.05), the proteins, except proteins with affinity to Neutravidin® agarose beads, commonly identified in both experiments were considered as Ef.LTA-BPs. 2.7. Immobilization of Ef.LTA on beads and pull-down assay

One milligram of Ef.LTA was conjugated with 500 ␮g of biotinNHS at room temperature for 4 h with gentle agitation. After conjugation, unbound biotin-NHS was separated and discarded by using PD-10 desalting column (GE Healthcare, Uppsala, Sweden). The biotinylated Ef.LTA (Ef.LTA-biotin) was quantified by measuring the phosphate contents as described previously (Baik et al., 2008).

The pull-down assay using Ef.LTA-immobilized beads was performed as described previously (Baik et al., 2013; Zuo et al., 2015). Briefly, after incubating the native beads (40 mg) or Ef.LTA-beads (40 mg) with recombinant human lipocalin-1 (1 ␮g) with gentle agitation at 4 ◦ C for 3 h, the interaction between Ef.LTA and lipocalin-1 was determined by Western blot analysis using antihuman lipocalin-1 antibody (Abcam, Cambridge, MA, USA).

2.3. Measurement of TLR2-binding activity

2.8. Measurement of biofilm formation

To assess the conjugation of Ef.LTA with biotin-NHS, CHO/CD14/TLR2 cells (2 × 106 cells/ml) were treated with Ef.LTA or Ef.LTA-biotin (0–10 ␮g/ml) at 37 ◦ C for 30 min. After a washing step, the cells were treated with streptavidin-FITC (Biolegend, San Diego, CA, USA) at room temperature for 10 min. Then, the binding of streptavidin-FITC to Ef.LTA-biotin on CHO/CD14/TLR2 cells was analyzed by flow cytometry using a FACSCalibur flow cytometer with CellQuest software (BD Biosciences, San Jose, CA, USA).

E. faecalis was grown in brain-heart infusion broth (BD Biosciences, Franklin, NJ, USA) in the presence or absence of human hemoglobin (Sigma-Aldrich) and recombinant human lipocalin-1 at 37 ◦ C for 24 h. Then, non-adherent cells were removed by washing with PBS and the remaining cells were stained with 0.1% crystal violet for 30 min. After washing with PBS, the stained crystal violet was solubilized with 95% ethanol containing 0.1% acetic acid (v/v) for the measurement of optical density at 595 nm with the VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA).

2.4. Determination of nitric oxide (NO) and macrophage inflammatory protein-1˛ (MIP-1˛)

2.9. Statistical analysis The induction of NO and MIP-1␣ by Ef.LTA or Ef.LTA-biotin in RAW 264.7 cells was determined as described previously (Lee et al., 2015).

The mean values ± standard deviations were determined from triplicate samples. A two-tailed t-test was used to determine sta-

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Fig. 1. A schematic experimental strategy for the identification of human salivary Ef.LTA-BPs.

tistical significance. Differences between the experimental groups and non-treatment control group were considered statistically significant at P < 0.05.

3. Results 3.1. Biotinylated Ef.LTA retained its binding capacity for TLR2 and CD14

biotinylation of Ef.LTA, the interaction between FITC-conjugated streptavidin and Ef.LTA-biotin was assessed on CHO/CD14/TLR2 cells co-expressing TLR2 and CD14, which are known receptors for Ef.LTA (Park et al., 2013). Fig. 2A indicates that streptavidin-FITC interacted with Ef.LTA-biotin-treated CHO/CD14/TLR2 cells but not with non-biotinylated Ef.LTA-treated cells (Fig. 2B). These results suggest that Ef.LTA was successfully biotinylated.

3.2. Ef.LTA-biotin retained its immuno-stimulatory activities In prior to isolation of salivary Ef.LTA-BPs, Ef.LTA-biotin was prepared using amine-reactive biotin-NHS (Fig. 1). Using a phosphate assay, the yield of the obtained Ef.LTA after biotinylation was determined to be 81% (data not shown). To confirm the

The immuno-stimulatory potential of Ef.LTA to induce NO and MIP-1␣ in a macrophage cell-line, RAW 264.7, has been reported in our previous studies (Baik et al., 2008; Park et al., 2013). To

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Fig. 2. Biotinylation did not alter the biological properties of Ef.LTA. (A–B) CHO/CD14/TLR2 cells were treated with (A) biotinylated Ef.LTA (Ef.LTA-biotin) or (B) Ef.LTA for 30 min. At the end of incubation, the cells were treated with streptavidin-FITC for 10 min at room temperature. Binding of streptavidin to Ef.LTA-biotin was analyzed by flow cytometry. The filled histogram represents the non-treatment group. Values indicate the percentage of cells exhibiting fluorescence. One representative result among three similar results is shown. (C–D) RAW 264.7 cells were pretreated with recombinant mouse IFN-␥ for 1 h, followed by stimulation with Ef.LTA-biotin or Ef.LTA for 24 h. The culture media were then analyzed for (C) NO and (D) MIP-1␣ production. Bars indicate mean values ± standard deviations. An asterisk (*) signifies statistical significance at P < 0.05 compared to the non-treatment (NT) control group. The data represent one of three representative results.

confirm the biological activities of the Ef.LTA-biotin, RAW 264.7 cells were treated with Ef.LTA-biotin and the production of NO and MIP-1␣ was measured. The Ef.LTA-biotin augmented NO production (Fig. 2C) at 3 ␮g/ml to levels similar to those noted in the non-biotinylated Ef.LTA treatment group. Although NO production induced by Ef.LTA-biotin was decreased at 10 ␮g/ml, it could be that the biotin-bound D-alanine of Ef.LTA structure (Fig. 1) may lose the immunostimulating activities since D-alanine of LTA structure is important for the immunostimualting activities (Grangette et al., 2005). In addition, Ef.LTA-biotin increased the production of MIP1␣ (Fig. 2D) to levels similar to those found in the non-biotinylated Ef.LTA sample, suggesting that the Ef.LTA-biotin did not lose its biological activityafter the biotinylation.

3.3. Ef.LTA-BPs in the human saliva were identified To identify salivary Ef.LTA-BPs, Ef.LTA-biotin was incubated with human saliva and Ef.LTA-BPs were isolated using Neutravidin® agarose beads. After Ef.LTA-BPs were separated onto

12% SDS-PAGE and visualized by silver staining (Fig. 3), the proteins were identified using LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Among a total 23 identified proteins with statistical significance (P < 0.05) obtained from the independently performed experiments, six of them that were repetitively identified were considered as Ef.LTA-BPs including SPLUNC2, zymogen granule protein 16 homolog B (ZG16B), hemoglobin subunit ␣ and ␤, apolipoprotein A-I and lipocalin-1 (Table 1).

3.4. Validation of Ef.LTA-BPs with lipocalin-1 Among the identified Ef.LTA-BPs, we selected lipocalin-1 to validate the Ef.LTA-BP because the other Ef.LTA-BPs identified in this study have either been already defined as LTA-BPs or specific antibodies to them are not yet commercially available. Using a pull-down assay, we showed the interaction of Ef.LTA with recombinant human lipocalin-1 (Fig. 4), which was in agreement with the peptide identification data.

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Table 1 Ef.LTA-BPs identified in human saliva by mass spectrometry. Accession number

Protein description

Peptide sequence

Peptide score

IPI00304557

Short palate lung and nasal epithelium carcinoma-associated protein 2

ISNSLILDVK

78

IPI00060800

Zymogen granule protein 16 homolog B

IPI00410714

Hemoglobin subunit alpha

IPI00654755

Hemoglobin subunit beta

IPI00021841

Apolipoprotein A-I

IPI00009650

Lipocalin-1

IFIHSLDVNVIQQVVDNPQHK LLPTNTDIFGLK STVSSLLQK VDLGVLQK FVNSVINTLK LKVDLGVLQK LLNNVISK LGALGGNTQEVTLQPGEYITK

75 64 64 60 59 58 46 84

VSVGLLLVK YFSTTEDYDHEITGLR LDGQISSAYPSQEGQVLVGIYGQYQLLGIK VGAHAGEYGAEALER

56 42 39 82

VADALTNAVAHVDDMPNALSALSDLHAHK VNVDEVGGEALGR

40 85

LLVVYPWTQR LLDNWDSVTSTFSK QGLLPVLESFK AKPALEDLR WQEEMELYR AVLEKTDEPGKYTADGGK AMTVDREFPEMNLESVTPMTLTTLEGGNLEAK

53 73 49 44 41 83 41

Expected functions

Binds to LPS; Exhibits antibacterial activity and anti-inflammatory effects (Abdolhosseini et al., 2012; Ghafouri et al., 2004; Gorr et al., 2011)

Promotes oral bacterial adhesion; Inhibits TLR-mediated NF-␬B signaling (Ambatipudi et al., 2010; Park et al., 2011) Binds to LTA; Potentiate LTA-induced IL-6 production in macrophages (Hasty et al., 2006)

Binds to LTA; Protect against sepsis and acute lung injury by neutralizing LTA (Jang et al., 2012; Jiao and Wu, 2008) Inhibits bacterial growth by acting as a siderophore (Fluckinger et al., 2004)

Fig. 4. Recombinant human lipocalin-1 (rhLipocalin-1) directly interacted with Ef.LTA. Native beads (Beads; 40 mg) or Ef.LTA-beads (Ef.LTA-Beads; 40 mg) were incubated with rhLipocalin-1 (1 ␮g) at 4 ◦ C for 3 h. After washing with PBS, the interaction of Ef.LTA with rhLipocalin-1 was assessed by Western blot using anti-human lipocalin-1 antibody. The rhLipocalin-1 (1 ␮g, lane 3) was used as the positive control. One of the three representative results is shown. The predicted molecular mass (19 kDa) of rhLipocalin-1 is indicated by arrow.

effect (Fig. 5B). These results suggest that Ef.LTA-BPs are not always responsible for the biofilm formation of E. faecalis.

Fig. 3. The profile of human salivary Ef.LTA-BPs. The salivary proteins bound to Ef.LTA or Ef.LTA-biotin were isolated using Neutravidin® agarose beads (NA-agarose Beads). Then, the eluted proteins were subjected to SDS-PAGE followed by staining with silver nitrite. Ef.LTA were used as a negative control. One of two similar results is shown. SM; size marker.

3.5. Effect of Ef.LTA-BPs on the biofilm formation of E. faecalis To evaluate the function of Ef.LTA-BPs, the effect of hemoglobin and lipocalin-1 on E. faecalis biofilm formation was measured. Fig. 5A indicates that the treatment with hemoglobin enhanced biofilm formation of E. faecalis while lipocalin-1 did not have such

4. Discussion The interaction between salivary proteins and oral pathogens such as E. faecalis influences the ecological balance to favor or resist their invasion into the dentinal tubule, which might be associated with refractory apical periodontitis. It is, therefore, crucial to investigate the interaction of salivary proteins with Ef.LTA to gain an understanding of the progress of infectious diseases and host immunity during infection by E. faecalis in the oral cavity. In this study, we identified salivary proteins with high affinity to Ef.LTA using LTQ-Orbitrap FT mass spectrometry. Most of Ef.LTA-BPs, except hemoglobins which have been previously known as LTA-BPs responsible for potentiating LTA-induced

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Fig. 5. Hemoglobin, but not lipocalin-1, increased the biofilm formation of E. faecalis. E. faecalis was grown on 96-well culture plates for 24 h in the presence or absence of Ef.LTA-BPs, (A) hemoglobin (6.25, 12.5, 25, or 50 ␮g/ml) and (B) lipocalin-1 (62.5, 125, 250, or 500 nM). Then, biofilm formation was determined by crystal violet staining followed by spectrometric analysis at 595 nm with a microplate reader. Bars indicate mean values ± standard deviations. An asterisk (*) indicates statistical significance at P < 0.05 compared to the non-treatment (NT) control group. The data represent one of three representative results.

inflammation (Hasty et al., 2006), appear to be involved in antibacterial and/or anti-inflammatory properties implying that salivary Ef.LTA-BPs favorably facilitate the first line of host defense in oral cavity. SPLUNC2, mainly produced in the salivary gland, interacts with LPS resulting in anti-bacterial and anti-inflammatory activities (Abdolhosseini et al., 2012; Ghafouri et al., 2004; Gorr et al., 2011). Likewise, lipocalin-1, a secreted protein with binding affinity to lipophilic ligands including fatty acids, phospholipids, glycolipids and cholesterols (Glasgow et al., 1995), is also involved in the host defense against bacterial infection by acting as a scavenger molecule for microbial siderophores (Fluckinger et al., 2004). Apolipoprotein A-I is known as a major component of plasma and plays a protective role during sepsis and acute lung injury by neutralizing LTA (Jiao and Wu, 2008; Jang et al., 2012). ZG16B, a pancreatic adenocarcinoma upregulated factor (PAUF), could attenuate TLR2-mediated inflammatory cytokine production such as TNF-␣ by inhibiting NF-␬B activation via C-X-C chemokine receptor type 4 (CXCR4) signaling (Park et al., 2011). Among Ef.LTA-BPs, hemoglobin increased the biofilm formation of E. faecalis. This result is concordant with the previous reports showing that hemoglobin promotes the nasal colonization of Staphylococcus aureus (Pynnonen et al., 2011) and biofilm formation of Staphylococcus epidermidis (Dai et al., 2012). In addition, hemoglobin is known to enhance the binding of Candida albicans to the host extracellular matrix protein, fibronectin (Yan et al., 1996). Therefore, hemoglobin seems to be a positive regulator of bacterial adhesion and aggregation in the oral cavity. In contrast, lipocalin1 did not show any effect on the biofilm formation of E. faecalis. Therefore, the inhibitory effect on bacterial biofilm formation may not be a common feature of Ef.LTA-BPs. The salivary proteins including hemoglobin and SPLUNC2, which were previously identified as S. mutans LTA-binding proteins (Sm.LTA-BPs) in human saliva (Hong et al., 2014), were also captured in the present study. This similarity between Ef.LTA-BPs and Sm.LTA-BPs might be explained with broad spectrum of host to recognize the molecular pattern of bacterial component. Since LTA has been considered as a major microbe-associated molecular pattern of Gram-positive bacteria that is commonly composed of a glycolipid together with alditolphosphate-containing polymer (Percy and Grundling, 2014), the conserved structure of LTAs might affect the interaction of LTA-BPs with both LTAs. However, some profiles of Ef.LTA-BPs identified from present study are dissimilar to those of Sm.LTA-BPs probably due to the subtle differences of LTA structure between species or strains. For example, D-alanylation of LTA has been reported to be crucial for bacterial adhesion, biofilm

formation, and the interaction with opsonic antibodies and cationic antimicrobial peptides by affecting electrostatic force (Theilacker et al., 2006; Fabretti et al., 2006). However, previous structural analysis has demonstrated that the C-2 position of glycerolphosphate in Ef.LTA substituted with not only D-Ala but also unique substituents such as kojibiose and [D-Ala → 6]-␣-d-Glcp-(1 → 2-[D-Ala → 6]-␣d-GlcP-1 → ) (Theilacker et al., 2006). In addition, unlike most other LTAs that possess one or two saturated fatty acids, Ef.LTA is harboring one or two unsaturated fatty acids (Baik et al., 2011). Although further studies are required to investigate the key moiety of Ef.LTA for mediating the interaction between Ef.LTA and Ef.LTA-BPs, the structural difference between Ef.LTA and Sm.LTA might be responsible for the dissimilarity between Ef.LTA-BPs and Sm.LTA-BPs. It is notable that we captured previously known LTA-BPs including hemoglobins and apolipoprotein A-I, but a few other LTA-BPs such as soluble TLR2, CD14, and LBP were not detected in this study. These results would be worthy of speculating two possible explanation. First, the level of soluble TLR2 (Houssen et al., 2014; Zhao et al., 2014), CD14 (Uehara et al., 2003), and LBP (Froon et al., 1995) are lower in the saliva compared to those in serum. Second, the relatively higher level of apolipoprotein A (Ghafouri et al., 2003), hemoglobin (Huang, 2004), and lipocalin-1 (Gutierrez et al., 2013) might interfere with the interaction between Ef.LTA-biotin and the less abundant LTA-BPs. The use of biotinylated LTA has advantages in capturing LTA-BPs compared to those of LTA-immobilized beads. First, the biotinylated LTA is soluble, making it easier to assess the LTA-BPs than LTA-immobilized beads. For example, serum lipoproteins such as high-density lipoprotein or low-density lipoprotein have been found to bind soluble LTA with fast kinetics (Levels et al., 2003) leading to inhibition of LTA-mediated immune activation under non-immobilizing conditions (Grunfeld et al., 1999), but such effects was not shown under immobilized conditions (Bunk et al., 2010). Second, the use of biotinylated LTA is more sensitive than the use of LTA-immobilized beads as about six-fold less LTA is needed to isolate LTA-BPs (Jang et al., 2012). Third, it is specific because the two-step process using biotinylated Ef.LTA followed by capture with streptavidin-coupled beads efficiently limits non-specific binding. Since the interaction between salivary proteins and E. faecalis is crucial for regulation of homeostasis in oral environments, the identification of salivary components capable of interacting with virulence factors of E. faecalis is important for understanding the progress of apical periodontitis and host immunity in the oral cavity. In this study, we identified the salivary proteins with affinity for

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Ef.LTA that might provide valuable information for understanding the bacterial pathogenesis and host defense mechanisms during E. faecalis infection in the oral cavity.

Conflict of interest The authors have no conflicts of interest to declare.

Acknowledgements This work was supported by grants from the National Research Foundation of Korea, which is funded by the Korean government (NRF-2015R1A2A1A15055453 and NRF-2015M2A2A6A01044894) and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, which is funded by the Ministry of Health & Welfare (HI14C0469), Republic of Korea. We appreciate the Korea Basic Science Institute (KBSI) for providing the mass spectrometric analysis.

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