molecular oral microbiology molecular oral microbiology

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects S.W. Hong1,*, D.-G. Seo2,*, J.E. Baik1, K. Cho3, C.-H. Yun4 and S.H. Han1 1 Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, Seoul, Korea 2 Department of Conservative Dentistry and DRI, School of Dentistry, Seoul National University, Seoul, Korea 3 Division of Mass Spectrometry Research, Korea Basic Science Institute, Ochang, Korea 4 Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Korea

Correspondence: Seung Hyun Han, Department of Oral Microbiology and Immunology, DRI, and BK21 Plus Program, School of Dentistry, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul 110-749, Korea Tel.: + 82 2 740 8641; fax: + 82 2 743 0311; E-mail: [email protected] *These authors contributed equally to this work. Keywords: dental caries; lipoteichoic acid; salivary proteins; Streptococcus mutans Accepted 19 May 2014 DOI: 10.1111/omi.12057

SUMMARY Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to be an important cell-wall virulence factor involved in bacterial adhesion and induction of inflammation. Since Sm.LTA-binding proteins (Sm.LTA-BPs) might play an important role in pathogenesis and host immunity, we identified the Sm.LTA-BPs in the saliva of caries-free and caries-positive human subjects using Sm.LTAconjugated beads and LTQ-Orbitrap hybrid Fourier transform mass spectrometry. Sm.LTA was conjugated to N-hydroxysuccinimidyl-Sepharoseâ 4 Fast Flow beads (Sm.LTA-beads). Sm.LTA retained its biological properties during conjugation, as determined by the expression of nitric oxide and interferon-c-inducible protein 10 in a murine macrophage cell line and activation of Toll-like receptor 2 (TLR2) in CHO/CD14/TLR2 cells. Sm.LTA-BPs were isolated from pooled saliva prepared from 10 caries-free or cariespositive human subjects each, electrophoresed to see their differential expression in each group, and further identified by high-resolution 208

mass spectrometry. A total of 8 and 12 Sm.LTABPs were identified with statistical significance in the pooled saliva from the caries-free and cariespositive human subjects, respectively. Unique Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and b-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits a and b, prolactin-inducible protein, protein S100-A9, and SPLUNC2. Collectively, we identified Sm.LTA-BPs in the saliva of caries-free and caries-positive subjects, which exhibit differential protein profiles.

INTRODUCTION Streptococcus mutans is a Gram-positive facultative anaerobe that is a major causative bacterium for dental caries and the subsequent development of pulpal inflammation, such as pulpitis (Hamada & Slade, 1980; Hahn et al., 2000). Accumulating reports © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

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suggest that the virulence factors of S. mutans include lipoteichoic acid (LTA), antigen I/II, glucosyltransferase, glucan-binding proteins, and sugar transport systems (Hamada & Slade, 1980; Mitchell, 2003). Among these, LTA has been recognized as one of the major bacterial cell wall components involved in bacterial adhesion to dentin, bacterial resistance to antimicrobial agents such as antimicrobial peptides or antibiotics, and the induction of host immune responses leading to inflammation (Ginsburg, 2002). Lipoteichoic acid is composed of a hydrophobic glycolipid linked with an anionic polysaccharide, in which each moiety is responsible for interaction with LTAbinding proteins (LTA-BPs). For example, the glycolipid of LTA is essential for interaction with Toll-like receptor 2 (TLR2), leading to activation of immune responses, whereas the polysaccharide of LTA is recognized by carbohydrate-recognizing proteins, such as CD14, lipopolysaccharide (LPS)-binding proteins, L-ficolin and mannose-binding protein (Polotsky et al., 1996; Schroder et al., 2003; Lynch et al., 2004). LTABPs are involved in both bacterial pathogenesis and host immune responses to infection through such mechanisms as delivery of LTA to TLR2, induction of inflammation (Schroder et al., 2003), activation of complements (Lynch et al., 2004), and neutralization of LTA (Polotsky et al., 1996). Hence, the identification and characterization of LTA-BPs are crucial for understanding bacterial pathogenesis and host immune responses. Saliva plays an important role in the homeostatic regulation of pH, antimicrobial functions, food digestion and the formation of acquired pellicle (Bennick et al., 1983; Lenander-Lumikari & Loimaranta, 2000). Previous reports have shown that the characteristics of saliva, including the pH and profile of salivary proteins, alter remarkably during the progression of oral diseases such as dental caries, periodontitis and oral cancer (Henskens et al., 1996; Koscielniak et al., 2012; Jessie et al., 2013). For example, saliva from caries-positive subjects exhibits relatively acidic pH (Kuriakose et al., 2013) and different expression levels of proline-rich proteins, staterins, histatin, cystatin, lysozyme and lipocalins (Vitorino et al., 2006; Koscielniak et al., 2012). Although these alterations in saliva might be responsible for the progression of dental caries by affecting the function of proteins involved in bacterial killing, bacterial adhesion, or host immune © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

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responses (Bennick et al., 1983; Flo et al., 2004; Ganeshnarayan et al., 2012), the relevant molecular mechanisms have not been fully characterized. In the present study, we identified S. mutans LTA-BPs in two different types of saliva that had been obtained from caries-free or caries-positive subjects. METHODS Bacteria, reagents, and chemicals Streptococcus mutans (KCTC 3065) was obtained from the Korean Collection for Type Cultures (Daejon, Korea). Octyl-Sepharose CL-4B, DEAE-Sepharose and N-hydroxysuccinimidyl-Sepharoseâ 4 Fast Flow beads (NHS-beads) were purchased from SigmaAldrich (St Louis, MO). The synthetic lipopeptide, Pam2CSK4, was obtained from InvivoGen (San Diego, CA). Recombinant mouse interferon-c was purchased from R&D Systems (Minneapolis, MN). All other reagents were obtained from Sigma-Aldrich unless otherwise indicated. Purification of S. mutans LTA Highly-pure and structurally-intact LTA was isolated from S. mutans as described previously (Baik et al., 2008; Ryu et al., 2009; Hong et al., 2014). In brief, 100 g of S. mutans (wet weight) was suspended in 0.1 M sodium citrate buffer (pH 4.7), disrupted by ultrasonication and further extracted with n-butanol. Extracts were subjected to hydrophobic interaction column chromatography using Octyl-Sepharose followed by anion exchange column chromatography using DEAE-Sepharose. The amount of purified S. mutans LTA (Sm.LTA) was determined as dry weight. Conjugation of Sm.LTA with NHS-beads Five hundred milligrams of NHS-beads were washed with pyrogen-free water and then incubated with 2.5 mg of Sm.LTA with gentle agitation at 4°C for 4 h followed by further reaction with 0.5 M ethanolamine (pH 8.0) at 4°C for 1 h to block any remaining reactive sites on the NHS-beads. The native beads were prepared by single reaction with 0.5 M ethanolamine (pH 8.0) without Sm.LTA at 4°C for 1 h. After collecting the supernatant, the beads were washed with pyrogenfree water five times, and the conjugation of Sm.LTA 209

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to the beads (i.e. formation of Sm.LTA-beads) was confirmed by conducting Kaiser’s test (Kaiser et al., 1970). The amount of Sm.LTA bound to the beads was calculated by measuring the quantity of remaining Sm.LTA in the supernatant using a phosphate assay as described previously (Han et al., 2003). Analysis of production of inflammatory mediators The murine macrophage cell line, RAW264.7 (TIB71), was purchased from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco’s modified Eagle’s medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (Hyclone), 100 U ml 1 penicillin and 100 lg ml 1 streptomycin at 37°C in a humidified incubator with 5% CO2. The cells (1 9 106 cells ml 1) were plated and treated with various stimuli for 24 h in the presence of interferon-c. After 24 h, the culture supernatant was collected to measure inflammatory mediators. The accumulation of nitrite in culture supernatants was measured to determine nitric oxide (NO) production, as previously described (Green et al., 1982). NaNO2 was used as a standard for nitrite concentration and optical density at 540 nm was measured using a microtitre-plate reader (Molecular Devices, Sunnyvale, CA). The expression of interferon-c-inducible protein-10 (IP-10) was measured with a commercially available enzyme-linked immunosorbent assay kit (R&D Systems) according to the manufacturer’s instructions. Measurement of TLR2 activation To measure the activation of TLR2 by Sm.LTAbeads, the nuclear factor-jB (NF-jB) reporter cell line, CHO/CD14/TLR2, was used. Briefly, these cells constitutively co-express human CD14 and TLR2. Upon stimulation of TLR2, signaling through NF-jB induces cell surface expression of CD25 (Medvedev et al., 2001). The expression of CD25 on CHO/CD14/ TLR2 cells was measured by flow cytometry using a FACSCalibur flow cytometer with CELLQUEST software (BD Biosciences, San Jose, CA). Collection of saliva Collection of saliva samples was conducted with the approval of the Institutional Review Board of the 210

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Seoul National University Dental Hospital (IRB No. CRI11008). Ten caries-free and caries-positive subjects each were selected from outpatients. Outpatients showing good oral hygiene with no current caries or no more than two restorations for previous caries were categorized as caries-free subjects. Conversely, outpatients showing poor oral hygiene with more than three current caries or more than 10 restorations for previous caries were categorized as caries-positive subjects. The mean value  standard deviation (SD) for the Decayed–Missing–Filled Teeth index for caries-free and caries-positive subjects were 1.3  0.8 and 14  9.1, respectively. Ten caries-free and 10 caries-positive subjects fasted for at least 2 h before saliva collection and then brushed their teeth for 2 min without toothpaste. All individuals rinsed their mouths with water for 10 min, and then 10 ml of saliva was collected in 50 ml conical tubes. Proteins in the saliva were protected from degradation by adding Complete Mini Protease Inhibitor Cocktail EDTAfree tablets (Roche, Mannheim, Germany). Other non-protein components, such as cells, debris and insoluble materials were removed by centrifugation at 7000 g at 4°C for 15 min. After centrifugation, the supernatant was collected and stored at 80°C until use. To compare the protein profiles in saliva from caries-free and caries-positive subjects, the quantities of proteins in saliva samples were determined using a bicinchoninic acid assay kit (Pierce, Rockford, IL) and 10 lg of proteins from each sample were mixed with sample buffer [8% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol, 30% glycerol, and 0.02% bromophenol blue in 0.25 M Tris–HCl, pH 6.8] and boiled at 100°C for 10 min. The samples were loaded on 15% SDS–polyacrylamide gel electrophoresis (PAGE) gels, electrophoresed and stained with Coomassie blue. Isolation and identification of Sm.LTA-BPs One milligram of saliva proteins from each of 10 subjects from the caries-free or caries-positive group were pooled separately. After eliminating non-specifically bound proteins from each salivary pool by pre-incubation with native beads (300 mg) at 4°C for 1 h, the pre-cleared saliva was incubated with native beads (30 mg) or Sm.LTA-beads (30 mg) with gentle agitation at 4°C for 4 h. After washing the beads three times with phosphate-buffered saline, the bound proteins were eluted by boiling at 100°C for 10 min in © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

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the sample buffer and separated on 15% SDS–PAGE by electrophoresis at 120 V for 3 h. After visualizing the proteins by silver staining, the whole lanes containing visible and non-visible bands were carefully cut off and subsequently subjected to 7-Tesla Finnigan LTQ-Orbitrap hybrid Fourier transform mass spectrometry as previously described (Choi et al., 2011). The obtained mass and tandem mass spectra were analyzed with Mascot Daemon (Matrix Science, London, UK) using the IPI human database (IPI.HUMAN. v.3.73). The peptide score was 10 9 Log(P), where P indicates the probability that the observed match was a random event. Individual peptide scores over 36 were considered as identity or extensive homology (P < 0.05). The mass spectrometric analysis was performed three times independently, and proteins comprising more than one peptide with over 36 peptide scores in at least two independent analyses were identified as Sm.LTA-BPs.

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(P < 0.05) increased by stimulation with Sm.LTA or Sm.LTA-beads, whereas no such an induction occurred upon treatment with native beads (Fig. 1A, B). To further confirm the ability of Sm.LTA-beads to activate TLR2, CHO/CD14/TLR2 cells were stimulated with Sm.LTA-beads and various control groups including beads, Sm.LTA and Pam2CSK4, and TLR2-dependent CD25 expression was measured by A

B

Statistical analysis The mean values  SD were determined from triplicate samples, and a two-tailed t-test was used to determine statistical significance. Differences between test groups and non-treatment control groups were ascribed statistical significance when P < 0.05. All experiments, including mass spectrometric analyses, were conducted in triplicate under the same conditions.

C

RESULTS Sm.LTA-beads were prepared using NHS-beads To prepare Sm.LTA-beads, LTA purified from S. mutans was conjugated to NHS-beads, forming stable bonds with the primary amines of the D-alanine in Sm.LTA. The phosphate assay revealed that one milligram of beads contained approximately 6 lg of Sm.LTA after the conjugation (data not shown). The conjugated Sm.LTA retained immunostimulatory activity similar to that of free Sm.LTA To examine whether Sm.LTA retained its immunostimulatory activity after conjugation, RAW264.7 cells were stimulated with Sm.LTA-beads or native beads. The production of NO and IP-10 was significantly © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

Figure 1 Conjugated Streptococcus mutans lipoteichoic acid (Sm.LTA) induces inflammatory mediators. RAW264.7 cells (1 9 106 cells ml 1) were pretreated with interferon-c (0.1 ng ml 1) for 1 h, followed by stimulation with Sm.LTA-beads (0.15, 0.5, 1.5, or 5 mg ml 1), native beads (Beads, 5 mg ml 1), or Sm.LTA (30 lg ml 1) for 24 h. At the end of the incubation period, the culture supernatants were obtained for the analysis of (A) nitric oxide (NO) and (B) interferon-gamma-inducible protein-10 (IP-10) production. Data are mean value  SD. *Indicates experimental groups with statistical significance at P < 0.05 compared with the untreated group. (C) Conjugated Sm.LTA stimulates cells via Toll-like receptor 2 (TLR2). CHO/CD14/TLR2 cells (2 9 105 cells ml 1) were treated with Sm.LTA-beads (0.15, 0.5, 1.5, or 5 mg ml 1), native beads (Beads, 5 mg ml 1), Sm.LTA (30 lg ml 1), or Pam2CSK4 (0.1 lg ml 1) for 24 h. After incubation, CD25 expression induced by TLR2-dependent nuclear factor-jB activation was analyzed by flow cytometric analysis. The value in each histogram shows the percentage of CD25 expression. One of three similar results is shown.

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flow cytometry. The induction of CD25 expression was observed upon treatment with Sm.LTA-beads, Sm.LTA or Pam2CSK4, but not with native beads (Fig. 1C). These results suggest that Sm.LTA-beads retained the TLR2 dependent-immunostimulatory activity. The protein profiles of caries-free versus cariespositive saliva were modestly different To compare the protein contents of caries-free and caries-positive saliva, 10 lg of saliva from each subject was separated on 15% SDS–PAGE gel and stained with Coomassie blue to visualize the proteins. A

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As shown in Fig. 2, a similar pattern of salivary contents among caries-free individuals was observed (Fig. 2A); however, relatively irregular and different protein profiles were found for each individual saliva sample of the caries-positive group (Fig. 2B). Remarkably, we also found 1.7-fold higher protein concentrations in saliva from caries-positive subjects than in that from caries-free subjects (data not shown). Sm.LTA-BP profiles differ between caries-free and caries-positive saliva To isolate Sm.LTA-BPs, saliva from 10 individuals each from the caries-free and the caries-positive groups were pooled by group, and non-specifically

B

Figure 2 Protein profiles in human saliva. Ten micrograms of saliva samples from (A) caries-free subjects (#1 ~ 10) and (B) caries-positive subjects (#1 ~ 10) were loaded on 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and separated by electrophoresis. Separated proteins were visualized by Coomassie blue staining. SM, size marker.

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Figure 3 The protein profiles of Streptococcus mutans lipoteichoic acid binding proteins (Sm.LTA-BPs). Eluted samples from Sm.LTAbeads or native beads (Beads) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized by silver staining. One microgram of the saliva samples was loaded as a control. One of three similar results is shown. The filled triangle (►) indicates protein bands found at higher levels in caries-free samples than in caries-positive samples. The open triangle ( ) indicates protein bands found at higher levels in caries-positive samples than in caries-free samples.



© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

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Table 1 Streptococcus mutans lipoteichoic acid-binding proteins in caries-free saliva identified by high-resolution LTQ-Orbitrap hybrid Fourier transform mass spectrometry Protein description

Accession no.

Peptide sequence

Peptide score

Hemoglobin subunit b

IPI00654755

Prolactin-inducible protein

IPI00022974

Hemoglobin subunit a

IPI00410714

Histone H4

IPI00453473

Protein S100-A9

IPI00027462

Profilin-1

IPI00216691

Neutrophil defensin-1

IPI00005721

Short palate, lung and nasal epithelium carcinoma-associated protein 2

IPI00304557

VLGAFSDGLAHLDNLK VNVDEVGGEALGR SAVTALWGKVNVDEVGGEALGR VVAGVANALAHKYH FFESFGDLSTPDAVMGNPK EFTPPVQAAYQK LLVVYPWTQR LHVDPENFR TYLISSIPLQGAFNYK ELGICPDDAAVIPIK FYTIEILKVE IIIKNFDIPK VGAHAGEYGAEALER TYFPHFDLSHGSAQVK MFLSFPTTK VLSPADKTNVK ISGLIYEETR VFLENVIR TLYGFGG NIETIINTFHQYSVK LGHPDTLNQGEFK KDLQNFLK DSLLQDGEFSMDLR TFVNITPAEVGVLVGKDR IPACIAGER YGTCIYQGR LKVDLGVLQK LLPTNTDIFGLK LLNNVISK

98 94 84 84 82 55 52 36 79 71 65 54 82 67 44 43 74 48 43 63 52 44 83 40 66 44 54 51 42

binding proteins were removed by pre-incubation with native beads. After precipitating the Sm.LTA-BPs from each salivary pool, the Sm.LTA-BPs were separated by 15% SDS–PAGE and visualized by silver staining to see the distinct bands from each salivary pool (Fig. 3). Then, Sm.LTA-BPs in the whole lanes containing both visible and non-visible bands were identified using LTQ-Orbitrap hybrid Fourier transform mass spectrometry. A total of 77 and 57 proteins were initially identified in the saliva pool from cariesfree and carries-positive subjects, respectively (significantly different at P < 0.05). Among these proteins, 8 and 12 proteins that were identified more than twice in three independent experiments were selected and considered as Sm.LTA-BPs of caries-free saliva (Table 1) and caries-positive saliva (Table 2), respectively. Sm.LTA-BPs found in caries-free saliva included histone H4, profilin-1 and © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

neutrophil defensin-1, and those in caries-positive saliva included cystatin-C, cystatin-SN, cystatin-S, cystatin-D, lysozyme C, calmodulin-like protein 3 and b-actin. The Sm.LTA-BPs found in both groups were hemoglobin subunits a and b, prolactin-inducible protein, protein S100-A9 and SPLUNC2. DISCUSSION In the initiation and progression of dental caries, the interaction of salivary proteins with S. mutans, or specifically its virulence factor Sm.LTA, plays a crucial role in host immune responses and bacterial pathogenesis. Hence, the identification of Sm.LTABPs in saliva is important for understanding the pathogenesis of dental caries and host defense mechanisms, and for developing preventive and therapeutic agents against dental caries. In this 213

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Table 2 Streptococcus mutans lipoteichoic acid-binding proteins in caries-positive saliva identified by high-resolution LTQ-Orbitrap hybrid Fourier transform mass spectrometry Protein description

Accession no.

Peptide sequence

Peptide score

Prolactin-inducible protein

IPI00022974

Hemoglobin subunit b

IPI00654755

Short palate, lung and nasal epithelium carcinoma-associated protein 2

IPI00304557

Cystatin-C

IPI00032293

Cystatin-SN

IPI00305477

Hemoglobin subunit a

IPI00410714

Cystatin-S

IPI00032294

Lysozyme C

IPI00019038

Cystatin-D

IPI00002851

Calmodulin-like protein 3

IPI00216984

b-actin

IPI00021439

Protein S100-A9

IPI00027462

TYLISSIPLQGAFNYK TVQIAAVVDVIR ELGICPDDAAVIPIK FYTIEILKVE TFYWDFYTNR VLGAFSDGLAHLDNLK SAVTALWGKVNVDEVGGEALGR VVAGVANALAHKYH ISNSLILDVK AQEAEKLLNNVISK STVSSLLQK LKVDLGVLQK LLPTNTDIFGLK LVGGPMDASVEEEGVR RALDFAVGEYNK EEDRIIPGGIYNADLNDEWVQR ARQQTVGGVNYFFDVEVGR VGAHAGEYGAEALER TYFPHFDLSHGSAQVK MFLSFPTTK EENRIIPGGIYDADLNDEWVQR ALHFAISEYNK STDYGIFQINSR RLGMDGYR ATNYNAGDRSTDYGIFQINSR TLAGGIHATDLNDK SQPNLDNCPFNDQPK AADTDGDGQVNYEEFVR LSDEEVDEMIR SLGQNPTEAELR VFDKDGNGFVSAAELR SYELPDGQVITIGNER VAPEEHPVLLTEAPLNPK QEYDESGPSIVHR NIETIINTFHQYSVK LGHPDTLNQGEFK

90 83 68 65 55 86 69 55 82 80 58 56 54 111 51 65 48 80 70 48 60 58 85 52 46 105 61 85 58 48 41 100 51 47 76 53

study, we identified three classes of Sm.LTA-BPs: those associated with caries-free subjects, with caries-positive subjects, or with both (Table 3). Histone H4, profilin-1 and neutrophil defensin-1 were identified with high probability of protein matching in saliva from caries-free subjects, suggesting that these proteins are likely to be involved in antimicrobial host defense. Histone, an important component of chromatin, can directly bind to LTA and LPS, resulting in disruption of the bacterial membrane and a reduction of tumor necrosis factor-a and nitric oxide production, respectively (Augusto et al., 2003; Morita et al., 2013). 214

Neutrophil defensin-1 is a member of the a-defensin family, which exerts antimicrobial activity against a broad spectrum of microorganisms, such as bacteria, fungi and enveloped viruses, by disrupting the negatively charged membranes of microbes (White et al., 1995). Profilin-1 is a small ubiquitous protein involved in actin polymerization (Tobacman & Korn, 1982). In Listeria monocytogenes infections, profilin1 contributes to host defense by interfering with bacterial motility in the cytosol via its affinity to bacterial surface proteins (Grenklo et al., 2003). On the other hand, cystatins, lysozyme C, calmodulin-like protein 3, and b-actin were identified in © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

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Table 3 Classification of Streptococcus mutans lipoteichoic acid-binding proteins State

Protein name

Functions

References

Caries-free

Histone H4

Binds to Staphylococcus aureus lipoteichoic acid (LTA) and disrupts bacterial membranes Interferes with bacterial motility by interacting with actin monomer Disrupts microorganisms by interacting with negatively charged membranes Binds to hydroxyapatite and has antimicrobial effect on Aggregatibacter actinomycetemcomitans Inhibits immunostimulatory activity of lipopolysaccharide and hydrolyzes peptidoglycan Increases cell motility Interacts with LTA and interferes with bacterial entry Interacts with LTA and synergistically activates Toll-like receptor 2 (TLR2) and TLR4 Binds to various oral bacteria and inhibits bacterial colonization Regulates immune response via TLR4 and nuclear factor-jB pathway Has antimicrobial and anti-inflammatory functions by interaction with lipopolysaccharide

Augusto et al. (2003)

Profilin-1 Neutrophil defensin-1 Caries-positive

Cystatins

Lysozyme C

Calmodulin-like protein 3 Actin Both caries-free and caries-positive

Hemoglobin Prolactin-inducible protein Protein S100-A9 Short palate, lung and nasal epithelium carcinoma-associated protein 2

saliva from caries-positive subjects. These cariespositive-related proteins are likely associated with antimicrobial activity and cytoskeletal modulation. Cystatins, cysteine protease inhibitors, have antimicrobial activity against oral pathogens such as Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, leading to a decrease in their colonization (Ganeshnarayan et al., 2012). They are also involved in the formation of acquired pellicle through their interaction with hydroxyapatite (Johnsson et al., 1991). Lysozyme C hydrolyzes bacterial peptidoglycan and also counters LPS-induced inflammation by inducing a conformational change in LPS (Ohno & Morrison, 1989). Actin is a major cytoskeletal protein that composes actin filaments. It has been reported that the interaction of actin with LTA regulates bacterial entry into the cytosol (Sela et al., 2000). Calmodulin-like protein-3 is an epithelial-specific protein that is involved in migration of cells by stabilizing myosin-10 in oral epithelial cells, contributing to wound healing (Bennett et al., 2007). Hemoglobin subunits a and b, protein S100-A9, SPLUNC2, and prolactin-inducible protein were found in both caries-free and caries-positive saliva. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

Grenklo et al. (2003) White et al. (1995) Shomers et al. (1982), Ganeshnarayan et al. (2012) Takada et al. (1994)

Bennett et al. (2007) Sela et al. (2000) Hasty et al. (2006), Cox et al. (2012) Schenkels et al. (1997) Riva et al. (2012) Gorr et al. (2011)

Hemoglobin mainly exists in the blood but can also be found in the oral cavity in the case of leakage of gingival crevicular fluid or oral wounds (Marti et al., 2002; Fabian et al., 2008). Hemoglobin is known to synergistically up-regulate the immunostimulatory activity of LTAs of various Gram-positive bacteria by forming complexes with LTA in monocytes and macrophages (Hasty et al., 2006). In addition, its bactericidal activity against Gram-negative bacteria such as Escherichia and Salmonella species has also been reported (Hodson & Hirsch, 1958). Protein S100-A9 is an immunomodulative protein frequently found in the cytoplasm and/or nuclei of a wide range of cells, and the heterodimeric complex of protein S100-A9/S100-A8 has affinity to hydrophobic molecules (Kerkhoff et al., 1998). Recently, protein S100-A9 has been reported to be a TLR4 agonist, leading to inflammation via NF-jB activation (Riva et al., 2012). SPLUNC2 belongs to a class of bactericidal/permeability-increasing proteins and LPS-binding proteins exhibiting anti-inflammatory, antimicrobial and endotoxin neutralizing activities (Zhou et al., 2006). Prolactin-inducible protein is an LPS-binding protein capable of binding to many oral 215

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bacteria, including A. actinomycetemcomitans, P. gingivalis (Baik et al., 2013), Gemella haemolysans, Gemella morbillorium, Streptococcus acidominimus, Streptococcus oralis, Streptococcus salivarius and Streptococcus parasanguinis (Schenkels et al., 1997), and may play a role in the blockage of bacterial adhesion. Therefore, hemoglobin, protein S100-A9, SPLUNC2 and prolactin-inducible protein are believed to contribute to host innate immunity in the oral cavity. Saliva is critical in maintaining the homeostatic regulation of the oral environment, so interactions between salivary protein and microbial virulence factors might play a crucial role in host innate immunity. In this study, we identified and compared salivary proteins that interact with LTA of S. mutans, which is responsible for dental caries, using saliva from cariesfree and caries-positive subjects. These Sm.LTA-BP profiles could provide important clues to better understand the molecular interactions between the host and S. mutans during the progression of dental caries. ACKNOWLEDGEMENTS This work was supported by grants from the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (20100029116, 2008-0062421, 2012-009268) and the R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. REFERENCES Augusto, L.A., Decottignies, P., Synguelakis, M., Nicaise, M., Le Marechal, P. and Chaby, R. (2003) Histones: a novel class of lipopolysaccharide-binding molecules. Biochemistry 42: 3929–3938. Baik, J.E., Ryu, Y.H., Han, J.Y. et al. (2008) Lipoteichoic acid partially contributes to the inflammatory responses to Enterococcus faecalis. J Endod 34: 975–982. Baik, J.E., Hong, S.W., Choi, S. et al. (2013) Alphaamylase is a human salivary protein with affinity to lipopolysaccharide of Aggregatibacter actinomycetemcomitans. Mol Oral Microbiol 28: 142–153. Bennett, R.D., Mauer, A.S. and Strehler, E.E. (2007) Calmodulin-like protein increases filopodia-dependent cell motility via up-regulation of myosin-10. J Biol Chem 282: 3205–3212.

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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Molecular Oral Microbiology 29 (2014) 208–218

Differential profiles of salivary proteins with affinity to Streptococcus mutans lipoteichoic acid in caries-free and caries-positive human subjects.

Streptococcus mutans is a representative oral pathogen that causes dental caries and pulpal inflammation. Its lipoteichoic acid (Sm.LTA) is known to b...
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