In vivo modification of the enamel pellicle and saliva resveratrol levels after use of resveratrol-containing orodispersible capsules.

OBJECTIVES: To evaluate in vivo 1) the bioavailability of trans-resveratrol when administered through sublingual capsules; 2) the effect of resveratrol on the protein composition of the acquired enamel pellicle (AEP). DESIGN: Ten volunteers received a sublingual capsule containing 50 mg of trans-resveratrol. Unstimulated saliva was then collected after 0, 30, 60, and 120 min and AEP was collected after 120 min following administration of the capsule. In the next week, the volunteers received a placebo sublingual capsule, and saliva and AEP were collected again. Saliva samples were analyzed for free trans-resveratrol using high-performance liquid chromatopgraphy (HPLC), and AEP samples were subjected to proteomic analysis (nLC-ESI-MS/MS). RESULTS: Trans-resveratrol was detected in saliva at all the time points evaluated, with the peak at 30 min. A total of 242 proteins were identified in both groups. Ninety-six proteins were increased and 23 proteins were decreased in the Resveratrol group. Among the up-regulated proteins, isoforms of cystatins, PRPs, Mucin-7, Histatin-1, Lactotrasnferrin and Lysozyme-C were increased and the isoforms of Protein S100, Neutrophil defensins, Albumin, PRPs, and, Statherin were decreased in Resveratrol group. CONCLUSION: The sublingual capsule is effective at increasing the bioavailability of trans-resveratrol in saliva. Several proteins involved in important processes to maintain systemic and oral health homeostasis were identified. These proteins differently expressed due to the presence of trans-resveratrol deserve attention for future studies, since they have important functions, mainly related to antimicrobial action.

Proteomic profile of the acquired enamel pellicle of children with early childhood caries and caries-free children.

Acquired enamel pellicle plays an important role in the pathogenesis of early childhood caries (ECC), working as a protective interface between the tooth and the oral cavity. The aim of this cross-sectional in vivo proteomic study was to compare the acquired enamel pellicle protein profile of 3-5-year-old children with ECC (n = 10) and caries-free children (n = 10). Acquired enamel pellicle samples were collected and processed for proteomic analysis (nLC-ESI-MS/MS). In total, 241 proteins were identified. Basic salivary proline-rich protein 1 and 2, Cystatin-B, and SA were found only in the caries free group. When comparing caries free and ECC groups, lower protein levels were found in the caries free group for hemoglobin subunit beta, delta, epsilon, gamma-2, globin domain-containing protein and gamma-1, neutrophil defensin 3, serum albumin, protein S100-A8, and S100-A9. The proteins histatin-1, statherin, salivary acidic proline-rich phosphoprotein ½, proline-rich protein 4, submaxillary gland androgen-regulated protein 3B, alpha-amylase 1 and 2B were found at higher levels in the caries free group. The exclusive and the proteins found at higher levels in the caries free group might have protective functions that play a role in the prevention of caries, besides providing important insights to be evaluated in future studies for the possible development of new therapeutic strategies for ECC.

The abundance of lysozyme, lactoferrin and cystatin S in the enamel pellicle of children - Potential biomarkers for caries?

OBJECTIVE: In this study, the abundance of the protective salivary proteins lysozyme, lactoferrin, and cystatin S was quantified in the in situ formed pellicle of caries-free and caries-active children to determine whether they may be possible biomarkers for caries. DESIGN: Pellicle formation was performed in situ for 10 min on ceramic specimens from the oral cavity of children (5-8 years) with caries (n = 17) and without evidence of caries (n = 17). Additionally, unstimulated saliva was collected. Levels of lysozyme, lactoferrin, and cystatin S were measured in desorbed pellicle eluates and saliva using ELISA. RESULTS: No statistically significant differences were found in the occurrence of cystatin S and lysozyme in saliva and pellicle between caries-active and caries-free children. However, significantly higher amounts of lactoferrin were detected in the pellicle of caries-active children. CONCLUSION: The protective salivary protein lactoferrin may be a biomarker for caries susceptibility in children.

A review on the role of salivary MUC5B in oral health.

BACKGROUND: The salivary glycoprotein MUC5B plays a versatile role in maintaining oral health. It contributes to lubrication, pellicle formation, antimicrobial defense, and water retention, and its glycans are an important nutrient for oral bacteria. This review aimed to describe the role of MUC5B in oral health and examine changes in its levels and composition in cases of hyposalivation and xerostomia. HIGHLIGHT: In cases of hyposalivation, the reduction of total salivary MUC5B levels and MUC5B glycosylation patterns due to Sjögren's syndrome (SS) and medication intake appeared insignificantly limited. In patients with SS, xerostomia was related to reduced MUC5B levels at the anterior tongue. In cases of xerostomia, MUC5B glycosylation might be reduced, yet other factors such as total protein concentration, MUC7 levels and glycosylation, and salivary spinnbarkeit are involved. In contrast to SS- and medication-induced hyposalivation, radiotherapy in the head and neck region leads to a bona fide reduction in salivary MUC5B levels. CONCLUSION: Our findings suggest that MUC5B levels are clearly impaired in hyposalivation and xerostomia related to radiotherapy in the head and neck region versus those related to SS and medication intake. A reduction in glycosylation in the case of dry mouth appears associated with MUC5B and MUC7 as well as other factors.

The influence of fillers and protease inhibitors in experimental resins in the protein profile of the acquired pellicle formed in situ on enamel-resin specimens.

OBJECTIVE: This study evaluated the influence of the addition of fillers and/or protease inhibitors [(epigallocatechin gallate - EGCG) or (chlorhexidine - CHX)] in experimental resins in the protein profile of the acquired pellicle (AP) formed in situ on enamel-resin specimens. DESIGN: 324 samples of bovine enamel were prepared (6 × 6 × 2 mm). The center of each sample was added with one of the following experimental resins (Bis-GMA+TEGDMA): no filler, no inhibitor (NF-NI); filler no inhibitor (F-NI); no filler plus CHX (NF-CHX); filler plus CHX (F-CHX); no filler plus EGCG (NF-EGCG); filler plus EGCG (F-EGCG). Nine subjects used a removable jaw appliance (BISPM - Bauru in situ pellicle model) with 2 slabs from each group. The AP was formed for 120 min, in 9 days and collected with electrode filter paper soaked in 3% citric acid. The pellicles collected were processed for analysis by LC-ESI-MS/MS. RESULTS: A total of 140 proteins were found in the AP collected from all the substrates. Among them, 16 proteins were found in common in all the groups: 2 isoforms of Basic salivary proline-rich protein, Cystatin-S, Cystatin-AS, Cystatin-SN, Histatin-1, Ig alpha-1 chain C region, Lysozyme C, Mucin-7, Proline-rich protein 4, Protein S100-A9, Salivary acidic proline-rich phosphoprotein ½ and Statherin. Proteins with other functions, such as metabolism and transport, were also identified. CONCLUSION: The composition of the experimental resins influenced the protein profile of the AP. This opens a new avenue for the development of new materials able to guide for AP engineering, thus conferring protection to the adjacent teeth.

Proteomics of acquired pellicle in gastroesophageal reflux disease patients with or without erosive tooth wear.

OBJECTIVES: This in vivo study compared the protein profile of the acquired enamel pellicle (AEP) in volunteers 1) with gastroesophageal reflux disease (GERD) and erosive tooth wear (ETW) (BEWE ≥ 9; GE group); 2) with GERD without ETW (BEWE = 0; GNE group) and 3) control (without GERD and BEWE = 0; C group). MATERIALS AND METHODS: Twenty-four subjects (8/group) participated. AEP was formed during 120 min and collected. After protein extraction, the samples were submitted to reverse phase liquid chromatography coupled to mass spectrometry. Label-free proteomic quantification was performed using Protein Lynx Global Service software. RESULTS: In total, 458 proteins were identified. Seventy-six proteins were common to all the groups. The proteomic profile of the AEP was quite different among the distinct groups. The numbers of proteins exclusively found in the C, GE and GNE groups were 113, 110 and 81, respectively. Most of the proteins exclusively identified in the C and GNE groups bind metals, while those in the GE group are mainly membrane proteins. Many proteins were found exclusively in the reflux groups. In the quantitative analyses, when the GNE group was compared with the GE group, the proteins with the highest decreases were Lysozyme C, Antileukoproteinase, Cathepsin G, Neutrophil defensins and Basic salivary proline-rich proteins, while those with the highest increases were subunits of Hemoglobin, Albumin and isoforms of Cystatin. CONCLUSION: Profound alterations in the proteomic profile of the AEP were seen in GNE compared with GE volunteers, which might play a role in the resistance to ETW seen in the first. CLINICAL SIGNIFICANCE: This pioneer study compared the proteomic profile of the AEP of patients with GERD with or without ETW. Increased proteins in those without ETW might be protective and are good candidates to be added to dental products to protect against erosion caused by intrinsic acids.

The mucosal pellicle - An underestimated factor in oral physiology.

Bioadhesion and bio-adsorption of proteins, glycoproteins and other biomolecules are ubiquitous phenomena in the oral cavity. While the protective role of the adsorbed salivary biomolecules on teeth (the acquired enamel pellicle) is well established, it has yet to be defined whether comparable processes occur on the desquamating oral soft tissues. The general term for these layers is pellicle, but due to the different characteristics of the coated surfaces the enamel pellicle and mucosal pellicle are their own entities. There is considerable information on the enamel pellicle, whereas only limited data are available on the mucosal pellicle. This can be attributed to the difficult standardized preparation of this biological structure. Based on the present knowledge the abundant and characteristic components of the mucosal pellicle include secreted soluble mucins (MUC5B, MUC7), membrane-associated epithelial mucins (MUC1), and to a lesser degree CA VI, sIgA, and cystatin. However, it seems to be of completely different ultrastructure as compared with the enamel pellicle. Since it is comprised of larger glycoproteins retaining water, it might be considered as a hydrogel, and it appears to have a lower tenacity than the enamel pellicle. Maturation and turnover are influenced by the delivery of salivary proteins, by the flow of saliva and the underlying desquamating oral epithelium. Its probable functions include lubrication and moisture retention. In general, the mucosal pellicle can be regarded as an underestimated key player in oral physiology.

The proteomic profile of the acquired enamel pellicle according to its location in the dental arches.

OBJECTIVE: This study evaluated the variation in the protein profile of the acquired enamel pellicle (AEP) formed in vivo according to its location in the dental arches. DESIGN: The AEP was formed for 120min in 9 volunteers. Pellicle formed at upper+lower anterior facial (ULAFa; teeth 13-23 and 33-43), upper anterior palatal (UAPa; teeth 13-23), lower anterior lingual (LALi; teeth 33-43), upper+lower posterior facial (ULPFa; teeth 14-17 24-27, 34-37 and 44-47), upper posterior palatal (UPPa; teeth 14-17 and 24-27) and lower posterior lingual (LPLi; teeth 34-37 and 44-47) regions were collected separately and processed for analysis by label-free LC-ESI-MS/MS. RESULTS: Three-hundred sixty three proteins were identified in total, twenty-five being common to all the locations, such as Protein S100-A8, Lysozyme C, Lactoferrin, Statherin, Ig alpha-2, ALB protein, Myeloperoxidase and SMR3B. Many proteins were found exclusively in the AEP collected from one of the regions (46-UAPa, 33-LALi, 59-ULAFa, 31-ULPFa, 44-LPLi and 39-UPPa). CONCLUSIONS: The protein composition of the AEP varied according to its location in the dental arches. These results provide important insights for understanding the differential protective roles of the AEP as a function of its location in the dental arches.

Oral mucosal epithelial cells express the membrane anchored mucin MUC1.

OBJECTIVE: The presence of a stable salivary pellicle (SP) is essential to provide a wet surface for the oral mucosal epithelia. The oral mucosa is covered by the SP which is suggested to be a mixed film of both salivary and epithelial components. Our aim was to analyse the presence of membrane-anchored mucin MUC1 in the oral mucosal epithelia. DESING: The presence of MUC1 was studied by immunohistochemical and immunoelectron microscopical methods in 19 buccal mucosal specimens. The localization and intensity of the epithelial expression were analyzed. RESULTS: Strong staining of MUC1 was found in the epithelial cells of intermediate and superficial layers. Some basal cells were shown faint expression. In the intermediate and superficial layers, the MUC1 expression was seen mainly on the upper cell surface. Furthermore, the expression of MUC1 was noted in the cytoplasm near the nucleus and in the rough granules. By electron microscopy, extracellular domain of membrane-anchored molecules extruded about 15-30nm above the cell surface in the apical cells of the oral epithelium. Immunoelectron microscopic examination shows that MUC1 is mainly localized in the plasma membrane of epithelial cells and also in small vesicles (75-100nm) just below the plasma membrane. CONCLUSION: The membrane-anchored MUC1 is expressed in the superficial layer of the oral mucosal epithelium, especially on the upper surface of epithelial cells. MUCI may be the anchoring protein of the salivary pellicle stabilization.

Saliva and Serum Protein Exchange at the Tooth Enamel Surface.

The acquired enamel pellicle is an oral, fluid-derived protein layer that forms on the tooth surface. It is a biologically and clinically important integument that protects teeth against enamel demineralization, and abrasion. Tooth surfaces are exposed to different proteinaceous microenvironments depending on the enamel location. For instance, tooth surfaces close to the gingival sulcus contact serum proteins that emanate via this sulcus, which may impact pellicle composition locally. The aims of this study were to define the major salivary and serum components that adsorb to hydroxyapatite, to study competition among them, and to obtain preliminary evidence in an in vivo saliva/serum pellicle model. Hydroxyapatite powder was incubated with saliva and serum, and the proteins that adsorbed were identified by mass spectrometry. To study competition, saliva and serum proteins were labeled with CyDyes, mixed in various proportions, and incubated with hydroxyapatite. In vivo competition was assessed using a split-mouth design, with half the buccal tooth surfaces coated with serum and the other half with saliva. After exposure to the oral environment for 0 min, 30 min and 2 h, the pellicles were analyzed by SDS-PAGE. In pure saliva- or serum-derived pellicles, 82 and 84 proteins were identified, respectively. When present concomitantly, salivary protein adsorbers effectively competed with serum protein adsorbers for the hydroxyapatite surface. Specifically, acidic proline-rich protein, cystatin, statherin and protein S100-A9 proteins competed off apolipoproteins, complement C4-A, haptoglobin, transthyretin and serotransferrin. In vivo evidence further supported the replacement of serum proteins by salivary proteins. In conclusion, although significant numbers of serum proteins emanate from the gingival sulcus, their ability to participate in dental pellicle formation is likely reduced in the presence of strong salivary protein adsorbers. The functional properties of the acquired enamel pellicle will therefore be mostly dictated by the salivary component.

Structural modifications of the salivary conditioning film upon exposure to sodium bicarbonate: implications for oral lubrication and mouthfeel.

The salivary conditioning film (SCF) that forms on all surfaces in the mouth plays a key role in lubricating the oral cavity. As this film acts as an interface between tongue, enamel and oral mucosa, it is likely that any perturbations to its structure could potentially lead to a change in mouthfeel perception. This is often experienced after exposure to oral hygiene products. For example, consumers that use dentifrice that contain a high concentration of sodium bicarbonate (SB) often report a clean mouth feel after use; an attribute that is clearly desirable for oral hygiene products. However, the mechanisms by which SB interacts with the SCF to alter lubrication in the mouth is unknown. Therefore, saliva and the SCF was exposed to high ionic strength and alkaline solutions to elucidate whether the interactions observed were a direct result of SB, its high alkalinity or its ionic strength. Characteristics including bulk viscosity of saliva and the viscoelasticity of the interfacial salivary films that form at both the air/saliva and hydroxyapatite/saliva interfaces were tested. It was hypothesised that SB interacts with the SCF in two ways. Firstly, the ionic strength of SB shields electrostatic charges of salivary proteins, thus preventing protein crosslinking within the film and secondly; the alkaline pH (≈8.3) of SB reduces the gel-like structure of mucins present in the pellicle by disrupting disulphide bridging of the mucins via the ionization of their cysteine's thiol group, which has an isoelectric point of ≈8.3.

Identification of acid-resistant proteins in acquired enamel pellicle.

OBJECTIVES: This study characterized the proteome profile of the acquired pellicle formed in vivo on enamel. Changes in this proteome profile after exposure to lactic or citric acid were also evaluated. METHODS: Volunteers (n=8) were subjected to dental prophylaxis. After 2 h to allow the formation of the acquired pellicle, the teeth were isolated with cotton rolls and 1 mL of citric acid (1%, pH 2.5) or lactic acid (0.1 M pH 4.8) or deionized water was gently applied with a pipette on the anterior teeth (both maxillary and mandibular) for 10 s. In sequence, the pellicle was collected with an electrode filter paper soaked in 3% citric acid. This procedure was repeated for two additional days following a crossover protocol. Proteins were subjected to reverse phase liquid chromatography coupled to mass spectrometry (nLC-ESI-MS/MS). MS/MS data were processed and submitted to Proteome Discoverer software. Searches were done using SWISS-PROT and TrEMBL databases for human proteins. RESULTS: In total, seventy-two proteins were present in all groups and were submitted to quantitative analysis (SIEVE). Some of these proteins were increased more than two-fold after exposure to the acids. Among them, cystatin-B was increased 20- and 13-fold after exposure to citric and lactic acids, respectively. Additionally, some proteins were identified in only one of the groups (18, 5, and 11 proteins for deionized water, citric and lactic acids, respectively). CONCLUSIONS: Our results open new insights regarding potentially acid-resistant proteins that could be added to dental products to prevent acidic dissolution of the teeth.

What interactions drive the salivary mucosal pellicle formation?

The bound salivary pellicle is essential for protection of both the enamel and mucosa in the oral cavity. The enamel pellicle formation is well characterised, however the mucosal pellicle proteins have only recently been clarified and what drives their formation is still unclear. The aim of this study was to examine the salivary pellicle on particles with different surface properties (hydrophobic or hydrophilic with a positive or negative charge), to determine a suitable model to mimic the mucosal pellicle. A secondary aim was to use the model to test how transglutaminase may alter pellicle formation. Particles were incubated with resting whole mouth saliva, parotid saliva and submandibular/sublingual saliva. Following incubation and two PBS and water washes bound salivary proteins were eluted with two concentrations of SDS, which were later analysed using SDS-PAGE and Western blotting. Experiments were repeated with purified transglutaminase to determine how this epithelial-derived enzyme may alter the bound pellicle. Protein pellicles varied according to the starting salivary composition and the particle chemistry. Amylase, the single most abundant protein in saliva, did not bind to any particle indicating specific protein binding. Most proteins bound through hydrophobic interactions and a few according to their charges. The hydrophobic surface most closely matched the known salivary mucosal pellicle by containing mucins, cystatin and statherin but an absence of amylase and proline-rich proteins. This surface was further used to examine the effect of added transglutaminase. At the concentrations used only statherin showed any evidence of crosslinking with itself or another saliva protein. In conclusion, the formation of the salivary mucosal pellicle is probably mediated, at least in part, by hydrophobic interactions to the epithelial cell surface.

Proteome and peptidome of human acquired enamel pellicle on deciduous teeth.

Understanding the composition and structure of the acquired enamel pellicle (AEP) has been a major goal in oral biology. Our lab has conducted studies on the composition of AEP formed on permanent enamel. The exhaustive exploration has provided a comprehensive identification of more than 100 proteins from AEP formed on permanent enamel. The AEP formed on deciduous enamel has not been subjected to the same biochemical characterization scrutiny as that of permanent enamel, despite the fact that deciduous enamel is structurally different from permanent enamel. We hypothesized that the AEP proteome and peptidome formed on deciduous enamel may also be composed of unique proteins, some of which may not be common with AEP of permanent enamel explored previously. Pellicle material was collected from 10 children (aged 18-54 months) and subjected to mass spectrometry analysis. A total of 76 pellicle proteins were identified from the deciduous pellicle proteome. In addition, 38 natural occurring AEP peptides were identified from 10 proteins, suggesting that primary AEP proteome/peptidome presents a unique proteome composition. This is the first study to provide a comprehensive investigation of in vivo AEP formed on deciduous enamel.

Salivary proteins promote proteolytic activity in Streptococcus mitis biovar 2 and Streptococcus mutans.

A major function of the salivary pellicle on oral surfaces is to promote colonization of the commensal microbiota by providing binding sites for adherence. Streptococcus mitis is an early colonizer of the oral cavity whereas Streptococcus mutans represents a later colonizer. To survive and grow, oral bacteria produce enzymes, proteases and glycosidases, which allow them to exploit salivary proteins as a nutrient source. In this study, adherence and proteolytic activity of S. mitis biovar 2 and S. mutans were investigated in a flow-cell model in the presence of different populations of surface-associated salivary proteins. Streptococcus mitis biovar 2 adhered well to surfaces coated with both a MUC5B-enriched fraction and a pool of low-density proteins containing MUC7, amylase, cystatin, gp340, immunoglobulin A, lactoferrin, lysozyme and statherin, whereas adherence of S. mutans to these proteins was poor. In environments of MUC5B or the low-density proteins, both S. mitis biovar 2 and S. mutans showed high levels of proteolytic activity. For S. mitis in the MUC5B environment, most of this activity may be attributable to contact with the molecules in the fluid phase although activity was also enhanced by adherence to surface-associated MUC5B. These data suggest that although they differ in their capacity to adhere to surface-associated salivary proteins, in the natural environment exploitation of saliva as a nutrient source can contribute to survival and colonization of the oral cavity by both S. mitis biovar 2 and S. mutans.

The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces.

The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S. mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.

Human common salivary protein 1 (CSP-1) promotes binding of Streptococcus mutans to experimental salivary pellicle and glucans formed on hydroxyapatite surface.

The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 µg/mL). However, S. mutans cells exposed to rCSP-1 (10 µg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

Protein and mucin retention on oral mucosal surfaces in dry mouth patients.

Oral homeostasis depends largely on proteins and mucins present in saliva that coat all oral surfaces. The present study compared the protein composition of residual fluid on mucosal surfaces in subjects with normal salivary flow with that of patients with dry mouth caused by salivary hypofunction. Samples of residual mucosal fluid were collected using paper strips and then analysed by protein electrophoresis and immunoblotting. In both patients and controls, residual fluids on mucosal surfaces (except the anterior tongue in control subjects) had higher protein concentrations than unstimulated whole-mouth saliva. High-molecular-weight mucin (MUC5B) was present in greater amounts on the anterior tongue than on other surfaces in control subjects. In dry mouth patients who were unable to provide a measurable saliva sample, MUC5B was often still present on all mucosal surfaces but in reduced amounts on the anterior tongue. The membrane-bound mucin, MUC1, was prominent on buccal and labial surfaces in patients and controls. Statherin was still present on surfaces that were dried to remove salivary fluid, suggesting that it may be adsorbed as a protein pellicle. It is concluded that oral mucosal surfaces in dry mouth patients can retain MUC5B and other salivary proteins, although the functional integrity of these proteins is uncertain.

Role of lysine in interaction between surface protein peptides of Streptococcus gordonii and agglutinin peptide.

INTRODUCTION: Streptococcus gordonii interacts with the salivary pellicle on the tooth surface and plays an important role in dental biofilm formation. Reports show that the analog Ssp peptide (A11K; alanine to lysine at position 11 in the arranged sequence, (1)DYQAKLAAYQAEL(13)) of SspA and SspB of S. gordonii increased binding to the salivary agglutinin (gp-340/DMBT1) peptide (scavenger receptor cysteine-rich domain 2: SRCRP2). To determine the role of lysine in the binding of the Ssp(A11K) peptide to SRCRP2, we investigated whether an additional substitution by lysine influenced the binding of Ssp(A11K) peptide to SRCRP2 using a BIAcore biosensor assay. METHODS: Six analogs of the Ssp peptide with positive charges in surface positions on the structure were synthesized using substitution at various positions. RESULTS: The binding activity of analog Ssp(A4K-A11K) peptide was significantly higher than the other Ssp analogs. The binding activity rose under low ionic strength conditions. The distance between positively charged amino acids in the Ssp(A4K-A11K) peptide between 4K and 11K was 1.24 +/- 0.02 nm and was close to the distance (1.19 +/- 0.00 nm) between Q and E, presenting a negative charged area, on SRCRP2 using chemical computing graphic analysis. The molecular angle connecting 1D-11K-4K in the Ssp(A4K-A11K) peptide secondary structure was smaller than the other peptide angles (1D-11K-XK). The Ssp(A4K-A11K) peptide showed higher inhibiting activity for Streptococcus mutans binding to saliva-coated hydroxyapatite than the (A11K) peptide. CONCLUSION: The positioning of lysine is important for binding between Ssp peptide and SRCRP2, and the inhibiting effect on S. mutans binding to the tooth surface.

Adhesion of Streptococcus mutans to salivary proteins in caries-free and caries-susceptible individuals.

Adhesion of microorganisms to dental surfaces is the initial step in the formation of dental bacterial plaque. Streptococcus mutans (S. mutans) is considered the main causal agent of one of the most common diseases in humans: dental caries. Adherence of these bacteria results from the interaction of adhesins that form part of their structure with salivary components, specifically those that compose the acquired pellicle. The complexity of this interaction has been the subject of studies in past years, to the extent of identifying certain salivary components related to adhesion to enamel surfaces, such as proline-rich proteins (PRSs), Staherins, Histatins, Cystatins, etc. One of the objectives of this study was to determine the adhesion capacity of S. mutans to synthetic hydroxyapatite incubated with saliva samples of caries-active and caries-inactive individuals. For the purpose of these assays, both the whole saliva samples and the salivary protein extracts were used. They were obtained by separating the proteins contained in the simple SDS-PAGE, in three ranges of molecular weight, selected in accordance with the electrophoresis profile that was usually found. The results indicated that the adhesion of this microorganism was greater in caries-inactive patients, when tested with whole saliva and proteins in the 120-159 kDa molecular weight range. This suggests that adhesion, per se, does not have a definite effect on the mechanisms that cause the disease in some individuals. However, these are interesting findings that may contribute to the design of strategies to control the adhesion of S. mutans to the tooth's surface.