[Show abstract][Hide abstract] ABSTRACT: The acquired enamel pellicle (AEP) is important for minimizing the abrasion caused by parafunctional conditions as they occur for instance during bruxism. It is a remarkable feature of the AEP that a protein/peptide film can provide enough protection in normofunction to prevent teeth from abrasion and chipping. Despite its obvious critical role in the protection of tooth surfaces, the essential mechanical properties of the AEP with regard to this protective function have been poorly characterized. Objective: To measure the adhesion force between histatin 5, a primary AEP component, and hydroxyapatite (HA) surfaces. Methods: Both streptavidin-biotinylated histatin 5 and streptavidin-biotinylated human serum albumin were allowed to adsorb on a silica microsphere attached to an atomic force microscope (AFM) cantilever. A Multimode AFM was used to measure the adhesion force between the cantilevers containing protein-functionalized microspheres and the HA surface. All measurements were performed within a fluid cell containing water. The imaging was performed in tapping mode using a Si3N4 AFM cantilever while the adhesion forces were measured in AFM contact mode using the functionalized cantilever system. Results: A collection of force-distance curves (~3,000/replicate) was used to generate histograms that illustrate the adhesion force between the HA surface and histatins 5 or albumin. Histatin 5 (>1.830 nN) exhibited stronger adhesion forces to the HA surface than albumin (>0.282 nN). Conclusion: This study represents an objective approach to adhesion force measurements between histatin 5 and HA, and provides the experimental basis for measuring the same parameters for other AEP constituents. Such knowledge will help in the design of synthetic proteins and peptides with clinical benefits for protective functions of the enamel surface.
Support: NSERC grant #371813, CIHR grants #106657/#97577 CFI-LOF grant #25116. WLS is recipient of a CIHR New Investigator Award grant #113166, NIH/NIDCR/NIAID grants DE05672, DE07652, AI087803 and AI101067.
[Show abstract][Hide abstract] ABSTRACT: Candida albicans is the most pathogenic fungal species, commonly colonizing human mucosal surfaces. As a polymorphic species, C. albicans is capable of switching between yeast and hyphal forms, causing an array of mucosal and disseminated infections with high mortality. While the yeast form is most commonly associated with systemic disease, the hyphae are more adept at adhering to and penetrating host tissue and are therefore frequently observed in mucosal fungal infections, most commonly oral candidiasis. Objective:To evaluate the potential of Histatin 5 to protect the Human Oral Epithelium against C. albicans adhesion. Methods:Human Oral Epithelial Tissues (HOET) were incubated with PBS containing histatin 5 for 2 h, followed by incubation with C. albicans for 1 h at 37 °C. The tissues were then washed several times in PBS, transferred to fresh RPMI and incubated for 16 h at 37°C at 5 % CO2. HOET were then prepared for histopathological analysis using light microscopy. In addition, the TUNEL assay was employed to evaluate the apoptosis of epithelial cells using fluorescent microscopy. Proteins released from HOET and from C. albicans-incubated HOET were subjected to relative proteome quantification by mass spectrometry. Results:HOET preincubated with histatin 5 showed a lower rate of C. albicans growth and cell apoptosis when compared to the control groups (HOET alone and HOET incubated with C. albicans). The incubation of HOET with histatin 5 resulted in distinct proteome changes in the incubation buffer compared to both control groups. Conclusions:The data suggest that the coating with histatin 5 is able to reduce C. albicans colonization of epithelial cell surfaces and also protect the basal cell layers from undergoing apoptosis. Supported by:NIH/NIDCR/NIAID grants DE05672, DE07652, AI087803 and AI101067, FAPESP Grants#2011/23540-5, 2011/23543-4 and 2013/15412-2, NSERC grant#371813, CIHR grants#106657/#97577 and, CFI-LOF grant#25116. WLS is recipient of a CIHR New Investigator Award grant#113166.
[Show abstract][Hide abstract] ABSTRACT: The acquired enamel pellicle (AEP) is important for minimizing the abrasion caused by parafunctional conditions as they occur, for instance, during bruxism. It is a remarkable feature of the AEP that a protein/peptide film can provide enough protection in normofunction to prevent teeth from abrasion and wear. Despite its obvious critical role in the protection of tooth surfaces, the essential adhesion features of AEP proteins on the enamel surface are poorly characterized. The objective of this study was to measure the adhesion force between histatin 5, a primary AEP component, and hydroxyapatite (HA) surfaces. Both biotinylated histatin 5 and biotinylated human serum albumin were allowed to adsorb to streptavidin-coated silica microspheres attached to atomic force microscope (AFM) cantilevers. A multimode AFM with a Nanoscope IIIa controller was used to measure the adhesion force between protein-functionalized silica microspheres attached to cantilever tips and the HA surface. The imaging was performed in tapping mode with a Si3N4 AFM cantilever, while the adhesion forces were measured in AFM contact mode. A collection of force-distance curves (~3,000/replicate) was obtained to generate histograms from which the adhesion forces between histatin 5 or albumin and the HA surface were measured. We found that histatin 5 exhibited stronger adhesion forces (90% >1.830 nN) to the HA surface than did albumin (90% > 0.282 nN). This study presents an objective approach to adhesion force measurements between histatin 5 and HA, and provides the experimental basis for measuring the same parameters for other AEP constituents. Such knowledge will help in the design of synthetic proteins and peptides with preventive and therapeutic benefits for tooth enamel.
Journal of dental research 03/2014; · 3.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dental erosion is a multifactorial condition that can result in the loss of tooth structure and function, potentially increasing tooth sensitivity. The exposure of enamel to acids from non-bacterial sources is responsible for the progression of erosion. These erosive challenges are counteracted by the anti-erosive properties of the acquired pellicle (AP), an integument formed in vivo as a result of selective adsorption of salivary proteins on the tooth surface, containing also lipids and glycoproteins. This review provides an in-depth discussion regarding how the physical structure of the AP, along with its composition, contributes to AP anti-erosive properties. The physical properties that contribute to AP protective nature include pellicle thickness, maturation time, and site of development. The pellicle contains salivary proteins embedded within its structure that demonstrate anti-erosive properties; however, rather than individual proteins, protein-protein interactions play a fundamental role in the protective nature of the AP. In addition, dietary and synthetic proteins can modify the pellicle, enhancing its protective efficiency against dental erosion. The salivary composition of the AP and its corresponding protein-profile may be employed as a diagnostic tool, since it likely contains salivary biomarkers for oral diseases that initiate at the enamel surface, including dental erosion. Finally, by modifying the composition and structure of the AP, this protein integument has the potential to be used as a target-specific treatment option for oral diseases related to tooth demineralization.
Archives of oral biology 02/2014; 59(6):631-638. · 1.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The acquire enamel pellicle (AEP) is formed by the selective adsorption of salivary proteins onto the enamel. Fluoride in/on the enamel surface may modulate the AEP proteome and, thereafter, the AEP functions. Objective: This study aimed to investigate the effect of fluoride on the AEP proteome. In addition, this study pioneered the use of label-free quantitative proteomics to better understand the composition and function of AEP proteins. Method: Hydroxyapatite (G1), hydroxyapatite pre-treated with 5%NaF (G2) and fluorapatite discs (G3) were set as substrate for AEP development. Each group (n=6) was incubated with parotid secretion (100µg) for 2h, at 37°C, in order to mimic in vivo AEP formation. Pellicle proteins were eluted from the discs by their sonication with 50mM ammonium bicarbonate, pH 7.8. AEP samples were trypisinized and subjected to LC-ESI-MS/MS. In addition, the obtained MS/MS spectra were searched against human protein databases. Mass spectrometric data were validated by SDS-PAGE and Western-Blot analysis. Moreover, the surfaces were analyzed for wettability and elemental composition (X-ray photoelectron spectroscopy). Result: This novel survey lead to the identification of 39 AEP proteins in G1, 33 in G2 and 52 in G3. The most abundant proteins present in all groups included amylase, histatin1, acidic PRP1 and lysozyme. Relative proteomic quantification was carried out for the 17 proteins observed in all three groups. Interestingly, 15, 9 and 27 proteins exclusively identified in G1, G2 and G3, respectively, were associated with distinctive molecular functions. Conclusion: Fluoride in/on the substrate qualitatively and quantitatively modulates AEP formation, effects which in turn will likely impact the formation of oral biofilms. These results present novel insights into the architecture of the AEP following fluoride treatment. Support CIHR grant# 106657, grant# 97577 and grant# 113166) and CFI-LOF grant# 25116.
[Show abstract][Hide abstract] ABSTRACT: Objectives: The initial tooth integument formed on enamel surfaces is the acquired enamel pellicle (AEP) which is formed by the selective adsorption of oral fluid proteins and peptides. This is followed by selective binding of oral microorganisms. Our recent studies have identified the early microbial colonizers in the 0-6h time frame, using DNA-DNA hybridization checkerboard and HOMIM microarray technologies. The sequential and parallel pattern of AEP and biofilm formation, though, has not yet been characterized. Insights into the mechanistic driving forces of these processes are vital for understanding the transitions from physiological to pathological conditions. The objective of our studies is to understand the biochemical determinants that dictate protein as well as bacterial adsorption, and to correlate both processes to ultimately gain insights into the critical steps in the formation of mature dental biofilm.
Methods: Tooth integuments were collected at 0, 2, 4 or 6 hours after thorough cleaning of tooth surfaces. The proteins in the AEP samples are being quantitatively analyzed using label-free LC-ESI-MS/MS, whereas the microbial compositions were determined using the HOMIM microarray.
Results: Samples have been obtained from 14 participating orally healthy subjects. Our earlier data indicated that the bacterial binding for the first 6 hours of integument formation comprise predominantly the species of Gemella haemolysans, Streptococcus cristatus, S. oralis, S. parasanguis I and II (p<0.05) as well as S. anginosus and intermedius, S. mitis and S. Cluster I. Both the raw label free LC-ESI-MS/MS data and microbial results will be computed to evaluate and correlate the protein and species patterns of early biofilm development.
Conclusions: With the in vivo AEP/early biofilm collection techniques developed in our laboratories and novel sensitive quantitatition assays new insights will be gained into the interplay between protein/peptides and bacteria in early tooth integuments.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: To quantify the proteome composition of the GCF in periodontal health (HH) and in sites with different clinical conditions in chronic periodontitis (CP) subjects.
5 subjects with HH and 5 with CP were submitted to full-mouth periodontal examination, and GCF sampling. Sites in the CP group were classified and sampled as periodontitis (P, probing depth, PD>4 mm), gingivitis (G, PD≤3mm with bleeding on probing, BOP), and healthy sites (H, PD≤3mm without BOP). GCF proteins were subjected to liquid chromatography electrospray ionization mass spectrometry for identification, characterization and quantification.
230 proteins were identified; 145 proteins were detected in HH, 214 in P, 154 in G, and 133 in H. Four proteins were exclusively detected at HH, 43 proteins at P, 7 proteins at G, and 1 protein at H. Compared to HH group, 35 and 6 proteins were more abundant in P and G (p<0.001), respectively; and 4, 15 and 37 proteins were less abundant in P, G and H (p≤0.01), respectively.
There are marked differences in the GCF proteome according to disease profile. Comprehension of the role of the identified proteins in the etiopathogenesis of periodontal disease may lead to biomarkers definition.
PLoS ONE 01/2013; 8(10):e75898. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Acquired enamel pellicle (AEP) is a protein film that forms on the enamel surface of teeth by selective adsorption of proteins and peptides present in the mouth. This protein film forms the interface between enamel and the damage oral biofilm, which modulates the attachment of bacteria found in oral biofilm. The overall goal of this study was to gain insight into the biological formation of the human in vivo AEP. This study hypothesized that AEP is created by the formation of successive protein layers, which consist of initial binding to enamel and subsequent protein-protein interactions. This hypothesis was examined by observing quantitative and qualitative changes in pellicle composition during the first two hours of AEP formation in the oral cavity. Quantitative mass spectrometry approaches were used to generate an AEP protein profile for each time-point studied. Relative proteomic quantification was carried out for the 50 proteins observed in all four time-points. Notably, the abundance of important salivary proteins, such as histatin 1, decrease with increasing of the AEP formation, while other essential proteins such as statherin showed constant relative abundance in all time-points. In summary, this is the first study that investigates the dynamic process to the AEP formation by using proteomic approaches. Our data demonstrated that there are significant qualitative and quantitative proteome changes during the AEP formation, which in turn will likely impact the development of oral biofilms.
PLoS ONE 01/2013; 8(7):e67919. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
International Journal of Molecular Sciences 01/2013; 14(1):920-34. · 2.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein's function is the identification of its interaction partners. We investigated interactions among and functions of histatin 1 and the other proteins that are present in saliva by using high-throughput mass spectrometric techniques. This led to the identification of 43 proteins able to interact with histatin 1. In addition, we found that these protein–protein interactions protect complex partners from proteolysis and modulate their antifungal activity. Our data contribute significantly to characterization of the salivary interactome and to understanding the biology of salivary protein complexes.
[Show abstract][Hide abstract] ABSTRACT: Background: Saliva is supersaturated with respect to calcium and phosphate ions. Salivary ions may well play a role in the subsequent adsorption of proteins and consequently in the formation of the acquired enamel pellicle. Among several biological functions, the enamel pellicle forms a selectively permeable barrier that regulates demineralization processes. Aim: The aim of this study was to evaluate the importance of salivary proteins when adsorbed on enamel surface and the resultant protective effect against demineralization without the presence of salivary ions. Methods: Enamel surfaces were coated with whole saliva, parotid saliva, dialyzed whole saliva or dialyzed parotid saliva (molecular weight cutoff 1 kDa). Adsorption was allowed to proceed for a period of 2 h. Enamel specimens were then washed with deionized water and immersed into a demineralization solution of pH 4.5 for 12 days. This solution was used to measure the amount of calcium and phosphate released from enamel specimens after the demineralization period. Results: All coated specimen groups showed a significantly higher protection than those not coated with any type of saliva. In addition, undialyzed saliva (whole saliva and parotid saliva) was more effective in protecting the enamel against demineralization than dialyzed saliva. Conclusion: The present investigation indicates that the ionic composition of saliva can amplify the demineralization protection effect by reducing acid-induced enamel demineralization. Moreover, a protective effect of salivary proteins without presence of ions was demonstrated in this study.
Caries Research 10/2012; 47(1):56-62. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The acquired enamel pellicle (AEP) is a thin acellular film that forms on tooth surfaces upon exposure to the oral environment. It consists predominantly of salivary proteins, but also includes non-salivary-derived proteins, carbohydrates, and lipids. Since it is the interface between teeth and the oral environment, the AEP plays a key role in the maintenance of oral health by regulating processes including lubrication, demineralization, and remineralization and shaping the composition of early microbial flora adhering to tooth surfaces. Knowledge of the 3D structure of the AEP and how that correlates with its protective functions may provide insight into several oral pathological states, including caries, erosion, and periodontal disease. This review intends to update readers about the latest discoveries related to the formation, ultrastructure, composition, and functions of the AEP, as well as the future of pellicle research, with particular emphasis on the emerging role of proteomic and microscopy techniques in oral diagnosis and therapeutics.
Journal of dental research 09/2012; · 3.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study aimed to determine whether dental calculus formation is really higher among patients with chronic kidney disease undergoing hemodialysis than among controls. Furthermore, the study evaluated correlations between dental calculus formation and dental plaque, variables that are related to renal disease and/or saliva composition.
The Renal Group was composed of 30 patients undergoing hemodialysis, whereas the Healthy Group had 30 clinically healthy patients. Stimulated whole saliva and parotid saliva were collected. Salivary flow rate and calcium and phosphate concentrations were determined. In the Renal Group the saliva collection was carried out before and after a hemodialysis session. Patients from both groups received intraoral exams, oral hygiene instructions, and dental scaling. Three months later, the dental calculus was measured by the Volpe-Manhold method to determine the rate of dental calculus formation.
The Renal Group presented a higher rate of dental calculus formation (p < 0.01). Correlation was observed between rate of dental calculus formation and whole saliva flow rate in the Renal Group after a hemodialysis session (r = 0.44, p < 0.05). The presence of dental calculus was associated with phosphate concentration in whole saliva from the Renal Group (p < 0.05).
In conclusion, patients undergoing hemodialysis presented accelerated dental calculus formation, probably due to salivary variables.
[Show abstract][Hide abstract] ABSTRACT: Objective: This study evaluated the use of human saliva in a commercial test (Dengue NS1 Ag STRIP ,Bio-Rad)for the detection of dengue NS1 protein during the clinical phase of dengue virus (DENV) infection.
Method: Whole saliva sample from 20 patients with acute infection of one of the four dengue serotypes, detected by RT-PCR and/or virus isolation and 20 healthy subjects were collected and used in this study. 50 μl of whole saliva was added to the Dengue NS1 Ag STRIP for a immunochromatographic test.
Result: The sensitivity of the Dengue NS1 Ag STRIP to patients with acute infection of one of the four dengue serotypes was not detected. The specificity achieved 100% to healthy subjectes.
Conclusion: Our findings could not support the use this commercial available diagnostic tool based on the NS1 antigen detection for the diagnosis of acute DENV infection using human saliva.
[Show abstract][Hide abstract] ABSTRACT: With recent progress in the analysis of the salivary proteome, the number of salivary proteins identified has increased dramatically. However, the physiological functions of many of the newly discovered proteins remain unclear. Closely related to the study of a protein’s function is the identification of its interaction partners.
Objective: The aim of this study was to identify the heterotypic complexes between histatin 1 and other salivary proteins in saliva and to explore the biological roles of these newly salivary complexes.
Method: Classical protein-protein interaction methods such as co-immunopreciptation and pull-down assay in combination with mass spectrometry analysis were carried out to gain insight of the histatin 1 interactome. In addition, the biological functions of the salivary protein complexes identified were assessed with specific assays that relate to known functions attributed to these proteins (e.g. candida albicans killing assay)
Result: Our exploration led to the identification of 43 proteins able to interact with histatin 1 in saliva. Moreover, we found that these newly protein-protein interactions protect complex partners from proteolysis and modulate their antifungal activity.
Conclusion: This study is the first to identify salivary protein complexes by using mass spectrometry. Our data contribute significantly to characterization of the salivary interactome and to understanding the biology of salivary protein complexes.
[Show abstract][Hide abstract] ABSTRACT: Understanding the composition and structure of the Acquired Enamel Pellicle (AEP) has been a major goal in oral biology. Our lab has conducted studies using mass spectrometry and proteomics on the composition of AEP formed on permanent enamel. Our exhaustive exploration has provided a comprehensive identification of more than 100 proteins from AEP formed on permanent enamel. AEP formed on deciduous enamel has not been subjected to the same biochemical characterization scrutiny as those of permanent enamel, despite the fact that deciduous enamel is structurally different than permanent enamel. We hypothesized that the AEP proteome formed on deciduous enamel may also be composed of unique proteins, some of which may not be common to AEP proteome of permanent enamel.Objective: The aim of this project is to investigate the proteome of human acquired enamel pellicle on deciduous teeth. Method: Deciduous human teeth were incubated with 300ug of saliva at 22°C for 2 h. After the pellicle-formation period, tooth specimens were removed and rinsed under distilled water for 10 seconds to remove residual saliva. The in vitro AEP material formed on deciduous enamel was harvested with 3% citric acid and sonication for 5 minutes. AEP proteins were trypisinized and subjected to mass spectrometry analysis. Result: At least 132 proteins were found based on the identification of two or more different peptides from the same parent protein. The theoretical pI of these AEP proteins fell mostly with the acidic range indicating that they exhibit negative charges at neutral pH.Conclusion: This is the first study to provide a comprehensive investigation of AEP on deciduous enamel. The MS approaches used open new avenues for the characterization of AEP components facilitating structural and functional in vivo studies.
[Show abstract][Hide abstract] ABSTRACT: The limited number of treatments for oral candidiasis resulted in the emergence of azole-resistant C. albicans strains, thus enforcing the need for novel antifungal treatments. Although histatin 5 (H5) demonstrates antifungal activity, its inhibitory effect when adhered to oral surfaces, resembling conditions of the in vivo pellicle, remains unexplored. Objectives: We aim to investigate the antifungal effect of H5 when it is adhered to PMMA and hydroxyapatite surfaces throughout a series of time intervals. Methods: Our biofilm assay involved developing monolayer protein integument (either H5 or albumin) on hydroxyapatite and PMMA discs, introducing C. albicans to this model system, and counting the number of adhered cells, throughout time, using scanning electron microscopy. Results: H5-coated-PMMA had significantly reduced number of cells compared to albumin-coated-PMMA at 30, 90, and 1440 minutes (p<0.0001), with the number of cells decreasing significantly across 90 and 1440 minutes (p<0.0001). Similarly, H5-coated-hydroxyapatite had significantly less cells compared to the albumin-coated surface at 90 and 1440 minutes (p<0.0001), with the number of cells decreasing significantly across 30, 90, and 1440 minutes (p<0.0001). The antifungal effect of PMMA and hydroxyapatite adhered H5 was strongest at 24 hours of biofilm maturation. In addition, cocci cells colonized albumin-coated-PMMA, while dense hyphael networks predominately colonized albumin-coated-hydroxyapatite. Conclusion: H5 maintains its, time-dependent, antifungal activity when adhered as a monolayer protein to PMMA and hydroxyapatite. Furthermore, C. albicans morphological plasticity may be influenced by the surface available for albumin adhesion.