Effect of Osteopontin on the Initial Adhesion of Dental Bacteria

Article pubs.acs.org/jnp

Effect of Osteopontin on the Initial Adhesion of Dental Bacteria Sebastian Schlafer,†,‡ Rikke L. Meyer,†,§ Duncan S. Sutherland,† and Brigitte Stad̈ ler*,† †

The Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, 8000, Denmark Department of Dentistry, Aarhus University, Aarhus, 8000, Denmark § Department of Bioscience, Microbiology, Aarhus University, Aarhus, 8000, Denmark ‡

S Supporting Information *

ABSTRACT: Bacterial biofilms are involved in numerous infections of the human body, including dental caries. While conventional therapy of biofilm diseases aims at eradication and mechanical removal of the biofilms, recent therapeutic approaches target the mechanisms of biofilm formation and bacterial adhesion in particular. The effect of bovine milk osteopontin, a highly phosphorylated whey protein, on adhesion of Streptococcus mitis, Streptococcus sanguinis, and Actinomyces naeslundii, three prominent colonizers in dental biofilms, to saliva-coated surfaces was investigated. While adhesion of A. naeslundii was not affected by osteopontin, a strong, dose-dependent reduction in the number of adhering S. mitis was shown. No difference in bacterial adhesion was observed for caseinoglycomacropeptide, another phosphorylated milk protein. Osteopontin did not affect bacterial viability, but changed bacterial surface hydrophobicity, and may be suggested to prevent the adhesins of S. mitis from interacting with their salivary receptors. The antiadhesive effect of osteopontin may be useful for caries prevention.

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sanguinis, and Actinomyces naeslundii, was investigated. These organisms are among the earliest colonizers of the human tooth and can be detected in high numbers both in initial dental biofilm12−14 and on incipient carious lesions.15,16 Studying and preventing initial adhesion of these bacteria is highly relevant for caries prevention. Specifically, (i) the ability of S. mitis, S. sanguinis, and A. naeslundii to adhere to saliva-coated polystyrene was determined in the absence and presence of osteopontin or caseinoglycomacropeptide, a similar milk protein, under flow conditions, and (ii) osteopontin- and caseinoglycomacropeptide-induced changes to cell surface properties were investigated by measuring the hydrophobicity of the three bacterial species using the microbial adhesion to hydrocarbons (MATH) test.

acterial biofilms are involved in a large number of microbial infections of the human body.1−5 Biofilm buildup occurs in several successive steps, of which the first is the adhesion of planktonic bacteria to a host surface.6 While conventional therapy of biofilm-related disease aims at the mechanical removal or eradication of the bacteria in the biofilm, recent approaches have focused on the mechanisms of biofilm formation and, in particular, on bacterial adhesion as a therapeutic target.7 If bacterial adhesion to the substratum is prevented, colonization and biofilm formation will fail and the organisms will be removed from their habitat-to-be. Importantly, therapeutic strategies targeting adhesion are nonbactericidal and do not subject the organisms to a selective pressure that might induce resistance to the treatment, as is the case for several antibiotics.8 Our group has demonstrated recently that bovine milk osteopontin, a glycosylated and highly phosphorylated whey protein, destabilizes biofilms in a multispecies flow cell model of dental biofilm.9 Acid production by bacteria in dental biofilms is the cause of dental caries, probably the most widespread disease of man. The most common means of caries prevention is the mechanical cleaning of the tooth surface, but even the combined use of a toothbrush and interdental floss does not achieve complete biofilm removal.10,11 In protected areas, such as pits, fissures, and approximal spaces, the biofilm remains undisturbed, and these niches are highly prone to the development of carious lesions. Targeting bacterial adhesion to saliva-coated surfaces is therefore a promising therapeutic approach for caries control. In the present study, the effect of osteopontin on the initial adhesion of three oral bacteria, Streptococcus mitis, Streptococcus © 2012 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Streptococcus mitis, Streptococcus sanguinis, and Actinomyces naeslundii are prominent colonizers of saliva-coated tooth surfaces and frequently isolated from both initial dental biofilm and incipient carious lesions.12−16 Our group employed these organisms recently in a five-species model of dental biofilm17 and demonstrated that osteopontin reduces biofilm growth and changes the species composition in the model.9 While the relative abundance of S. sanguinis and A. naeslundii increases in the presence of this protein, the proportion of S. mitis, the best biofilm former in the model, is reduced. In the present investigation, the effect of osteopontin on the initial adhesion of Received: July 31, 2012 Published: November 20, 2012 2108

dx.doi.org/10.1021/np300514z | J. Nat. Prod. 2012, 75, 2108−2112

Journal of Natural Products

Article

S. mitis, S. sanguinis, and A. naeslundii to saliva-coated polystyrene flow cells was determined. A marked effect of osteopontin on the adhesion of S. mitis was observed. Saliva coating of polystyrene was confirmed by QCM-D (Δf = −26.7 ± 0.6 Hz, ΔD = 2.4 ± 0.3 × 10−6). The presence of 26.5 μM osteopontin in the flow medium lowered the amount of adhered cells by approximately 80% (Figure 1a).

per area was not correlated with the amount of osteopontin present in the medium (Figure 1), as assessed via linear regression (R2 = 0.82). This finding makes osteopontin particularly interesting as a natural product to be used in caries prevention. Many other natural compounds considered in this context are predominantly derived from plants, e.g., from cranberry,18−20 and can potentially exhibit antimicrobial effects.21,22 Streptococcus sanguinis SK150 was not able to attach to the saliva-coated surface, irrespective of the presence of osteopontin (Supporting Information, Video S1). The organism also failed to form stable monospecies biofilms in the flow cells (data not shown), which suggests that successful colonization by S. sanguinis SK150 is dependent on interspecies coaggregation with other organisms. A. naeslundii AK6, the third organism employed in the present study, adhered well to the saliva-coated substrate and formed typical branched microcolonies in the flow cells (Figure 2a). Interestingly, the number of attached cells per area was not

Figure 1. Adhesion of Streptococcus mitis: (a) Fluorescent microscopy images of live/dead stained S. mitis SK24 attached to saliva-coated polystyrene surfaces in the absence (left) and presence of osteopontin (26.5 μM; right). Viable cells appear green; dead cells appear red. (b) Number of dead and viable S. mitis on 0.144 mm2 (red and green bars, respectively) attached to saliva-coated surfaces in the absence or presence of different concentrations of osteopontin or caseinoglycomacropeptide. THB = Todd-Hewitt broth. Error bars indicate standard deviations (*p < 0.01, ***p < 0.002, ****p < 0.00002). Figure 2. Adhesion of Actinomyces naeslundii: (a) Fluorescent microscopy images of live/dead stained A. naeslundii AK6 attached to saliva-coated polystyrene surfaces in the presence of osteopontin (26.5 μM). (b) Number of dead and live A. naeslundii on 0.144 mm2 (red and green bars, respectively) attached to saliva-coated surfaces in the absence or presence of different concentrations of osteopontin or caseinoglycomacropeptide. THB = Todd-Hewitt broth. Error bars indicate standard deviations. No statistically significant differences between different treatments were observed.

The effect of this protein on cell adhesion was dose dependent. A 10-fold lower osteopontin concentration (2.7 μM) still yielded ca. 80% fewer adhered cells. About 60% fewer cells adhered at 1.5 μM osteopontin, while no effect of this compound was seen for an osteopontin concentration of 0.6 μM. Importantly, no effect on bacterial adhesion could be observed when caseinoglycomacropeptide, another phosphorylated milk glycoprotein, was added to the medium in the same molar concentration as the highest concentration of osteopontin tested, which suggests a specific effect of the latter protein (Figure 1b). The impaired adhesion of S. mitis SK24 is not due to an antibacterial effect of osteopontin. It has been previously shown that the growth of S. mitis SK24 in planktonic culture is not affected by the presence of 26.5 μM osteopontin.9 Moreover, dead cells, stained red by BacLight, were among those that adhered to the saliva-coated surface, and the ratio of dead cells

significantly lower in the presence of 26.5 μM caseinoglycomacropeptide or osteopontin (Figure 2b), implying that the observed antiadhesive effect of the latter compound was specific for S. mitis. The mechanisms of bacterial adhesion to saliva-coated surfaces are complex and manifold. Considerable evidence shows that mitis-group streptococci adhere to salivary glycoprotein receptors via proteins of the antigen I/II 2109



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family,23,24 and binding of streptococcal surface proteins to salivary alpha-amylase is also well documented.25−27 Type I fimbriae play an important role for the adhesion of A. naeslundii to proline-rich proteins and statherin in saliva films,28−31 but very little data is available on fimbrial extensions in mitis-group streptococci and their possible role in mediating adhesion.32 In an attempt to explain the adhesive behavior of the three different bacterial species, changes in cell surface hydrophobicity were studied when exposed to osteopontin or caseinoglycomacropeptide. The MATH test33 showed that osteopontin interacted with the surfaces of all three organisms and rendered them more hydrophilic (Figure 3). Compared to

Article

EXPERIMENTAL SECTION

Materials. Phosphate-buffered saline (PBS) and toluene were purchased from Sigma (Brøndby, Denmark). Stimulated saliva was collected from healthy donors and prepared according to the method of de Jong et al.38 Ultrapure water (Millipore Corporation, Billerica, MA, USA) was used for all the experiments. Blood agar plates were purchased from SSI (Copenhagen, Denmark). Todd-Hewitt broth (THB) was obtained from Roth (Karlsruhe, Germany). Transparent polystyrene flow cells (μ-Slide VI0.4) were obtained from Ibidi GmbH (Planegg/Martinsried, Germany). BacLight bacterial viability stain was purchased from Invitrogen (Naerum, Denmark). n-Hexadecane (for synthesis) was obtained from Merck (Darmstadt, Germany). Osteopontin and caseinoglycomacropeptide were provided by Arla (Viby, Denmark). Osteopontin (also known as bone sialoprotein I) was first purified by Franzen et al. from bovine bone in 1985,39 and in 1989 and 1993 from human40 and bovine milk,41 respectively. The average MW of the compound used was 23.3 kDa, and it had a purity of 78%. The purity was taken into consideration when calculating the concentrations. Caseinoglycomacropeptide was first isolated by Jolle et al. in 1959.42 The average MW of this protein is 7500 Da, and it had a purity of 97−98%. Above pH 6, the protein is predominantly in its monomeric form.43 Quartz Crystal Microscopy with Dissipation Monitoring (QCM-D). QCM-D measurements (Q-sense E4, Gothenburg, Sweden) were used to analyze the adsorption of saliva to polystyrene-coated surfaces and the subsequent adsorption of osteopontin. Gold crystals (QSX 301, Q-sense, Gothenburg, Sweden) were coated with polystyrene by spin coating a 4 wt % polystyrene solution in toluene (25 s, 3000 rpm) followed by curing on a hot plate (5 min, 90 °C). Unless otherwise mentioned, the mass and dissipation measurements were monitored at 23 ± 0.02 °C, and PBS was used for all the experiments. When a stable baseline in the buffer solution was achieved, saliva (2:1 diluted in PBS) was introduced into the measurement chamber and left to adsorb until the surface was saturated. Then, the chamber was rinsed with buffer. The saliva-coated surface was than exposed to osteopontin (2 g L−1 in PBS). Upon surface saturation, the chamber was rinsed with buffer solution. The changes in frequency (Δf) and dissipation (ΔD) of the quartz crystal upon adsorption were monitored. Bacterial Strains. Human oral isolates S. mitis SK24, S. sanguinis SK150, and A. naeslundii AK6 were used for the experiments.17 16S rRNA sequence data have been deposited in GenBank (accession numbers: HQ219654, HQ219656, and HQ219658).44 The organisms were cultured aerobically on blood agar plates and grown at 35 °C in THB until late exponential phase before being used in the experiments.17 Flow Cell Adhesion Experiments. Liquid bacterial cultures were transferred to 100 mL of THB, THB containing 0.9 g L−1 osteopontin (26.5 μM), or THB containing 0.18 g L−1 (26.5 μM) caseinoglycomacropeptide and grown overnight at 35 °C on a rotary shaker (120 rpm; Buch & Holm, Herlev, Denmark). Bacterial suspensions were then diluted 1/10 with Milli-Q water, Milli-Q water containing 0.9 g L−1 osteopontin, or Milli-Q water containing 0.18 g L−1 caseinoglycomacropeptide, and optical density was adjusted to 0.05 (550 nm). The two tested proteins were added at approximately the same molar concentration. Bacterial adhesion was studied in polystyrene flow channels that allow direct microscopic analysis. Flow channels were conditioned with PBS and then with sterile saliva (2:1 diluted in PBS) for 30 min, followed by 30 min of PBS flow. Subsequently, bacterial suspensions were run through the flow channels (250 μL min−1; 28.3 mm min−1) for 3 h at 35 °C (Scheme 1). Then, the flow was halted, and the flow channels were washed with PBS and stained with BacLight live/dead stain according to the manufacturer’s instructions. In additional experiments, bacterial suspensions containing 0.02 g L−1 (0.6 μM), 0.05 g L−1 (1.5 μM), or 0.09 g L−1 (2.7 μM) osteopontin were inoculated. All experiments were performed in triplicate and repeated on a separate occasion.

Figure 3. The hydrophobicity of Streptococcus mitis SK24, Streptococcus sanguinis SK150, and Actinomyces naeslundii AK6 in pure Todd-Hewitt broth (THB) and in THB containing either caseinoglycomacropeptide or osteopontin (26.5 μM) was assessed by the MATH test. Error bars indicate standard deviations (*p < 0.01, **p < 0.005).

the other two organisms, the initial hydrophobicity of S. sanguinis SK150 was much lower, which suggests a link between surface hydrophobicity and the ability to adhere to saliva-coated surfaces, as this organism adhered poorly. Exposure to caseinoglycomacropeptide caused a much smaller change in hydrophobicity for S. mitis SK24 and A. naeslundii AK6 than exposure to osteopontin and left S. sanguinis SK150 unaffected. While the MATH test is not able to fully explain the effect of osteopontin on adhesion of the three organisms to a salivacoated substrate, two hypotheses for the different behavior of S. mitis SK24 and A. naeslundii AK6 are plausible. Osteopontin might interact specifically with adhesins of S. mitis SK24 and prevent them from binding to their salivary receptors. Alternatively, this protein might not bind directly to bacterial adhesins but instead bind to other molecules and form a hydrophilic layer on the bacterial cell surface. QCM-D experiments showed that the binding of osteopontin to a saliva-coated substrate is limited (Δf = −14.5 ± 0.7 Hz, ΔD = (1.4 ± 0.9) × 10−6), as compared to other substrates.34 An osteopontin layer on the bacterial surface might thus act as a barrier with low affinity to saliva that prevents the adhesins of S. mitis from interacting with their salivary receptors.35,36 Unlike S. mitis, A. naeslundii expresses type I fimbriae that appear as long hydrophobic cell appendages37 and might transgress the osteopontin barrier to enable successful adhesion even in the presence of high concentrations of this compound. However, further studies are required to elucidate the detailed molecular mechanisms involved. 2110



Article

(Microbiology, Aarhus University, Denmark) and L. Jasulaneca (iNANO, Aarhus University, Denmark) for excellent technical support. This work was funded by the Danish National Advanced Technology Foundation through the ProSURF platform project (Protein-Based Functionalisation of Surfaces) and by the Carlsberg Foundation.

Scheme 1. Schematic Illustration of the Experimental Setup to Characterize the Initial Adhesion of Bacteria to SalivaCoated Surfaces in Broth Containing Osteopontin under Flow

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Microscopic Analysis. A wide-field microscope (Zeiss Axiovert 200 M, Jena, Germany) equipped with a 100 W high-pressure mercury lamp (HB103, Osram; Rümlang, Switzerland), a 63× objective (Plan Apochromat, Zeiss, Jena, Germany), and narrow band filter sets F11001 and F11-007 (AHF Analysentechnik, Tübingen, Germany) was used for microscopic analysis. In each flow channel, eight randomly selected fields of view were imaged with a digital camera (AxioCam MRm, Jena, Zeiss), and the bacteria were counted. Average and standard deviation of triplicates from two independent repeats were calculated and statistically analyzed using an unpaired t test. Microbial Adhesion to Hydrocarbon (MATH) Test. The MATH test characterizes the hydrophobic properties of the cell surface of microorganisms.33 Liquid bacterial cultures were washed twice (3000g, 5 min) and resuspended in PBS, PBS containing 0.9 g L−1 osteopontin (26.5 μmol L−1), or PBS containing 0.18 g L−1 (26.5 μmol L−1) caseinoglycomacropeptide. Optical density was adjusted to 0.4 (550 nm; OD1). Then, 1 mL of each suspension was mixed with 200 μL of n-hexadecane. The mixtures were vortexed for 1 min, and phases were left to separate before the optical density of the aqueous phase was measured again (OD2). Hydrophobicity was calculated using the following equation: MATH (%) = (OD1 − OD2)/OD1 × 100. The average and standard deviations of triplicates from two independent repeats were plotted and statistically analyzed using an unpaired t test.

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ASSOCIATED CONTENT

S Supporting Information *

Video S1: S. sanguinis SK150 fails to adhere to saliva-coated flow cells. After inoculation, bacterial cells were stained with BacLight, and a single field of view was imaged for 20 s. Cells of S. sanguinis SK150 are not attached to the substratum. Field of view size: 143 × 143 μm. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: +45 8715 6668. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank B. Nyvad (Department of Dentistry, Aarhus University, Denmark), L. Schauser (iNANO, Aarhus University, Denmark), and P. L. Wejse (Arla Foods amba, Viby, Denmark) for fruitful discussions, and T. Wiegers 2111



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Effect of Osteopontin on the Initial Adhesion of Dental Bacteria

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