Specific and Nonspecific Interactions between Salivary Proteins and

Chapter 16

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Specific and Nonspecific Interactions between Salivary Proteins and Streptococcus mutans Chun-Ping Xu,1 Henk J. Busscher,1 Henny C. van der Mei,1 and Willem Norde*,1,2 1Department

of Biomedical Engineering, University Medical Center Groningen, and University of Groningen, Antonius Deusinglaan 1, 9713 AV, Groningen, The Netherlands 2Laboratory of Physical Chemistry and Colloid Science, Wageningen University and Research Center, Dreijenplein 6, 6703 HB, Wageningen, The Netherlands *E-mail: [email protected]

Adhesion of proteins to natural surfaces, such as the bacterial cell wall, may be controlled by non-specific and/or specific interactions. The latter ones are mediated by adhesins on the (bacterial) surface. This paper reports on the interaction between saliva proteins and Streptococcus mutans, an oral bacterium that is involved in tooth decay, by comparing two strains of S. mutans, one with and one without the adhesin antigen I/II on its surface. Bacterium-saliva interaction is characterized in terms of enthalpy (calorimetry), strength of adhesive bond (AFM), affinity (adsorption isotherms), and kinetics of adhesion of the bacterial strains to a saliva-coated surface (parallel plate flow chamber). The study shows that the presence of antigen I/II at the streptococcal surface adds favorable binding sites that are biologically recognized by (a number of) salivary proteins. Thus, superimposed on generic interactions, specific pH-dependent short-range interactions contribute dominantly to the adhesion of antigen I/II containing S. mutans to salivary coatings, such as salivary conditioning films on tooth surfaces.

© 2012 American Chemical Society In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Introduction Adhesion of bacteria is occurring widely spread in nature as well as in technological applications. In nature bacterial mass predominantly exists in biofilms at surfaces (1), as, e.g., in soil and surface waters, in the marine environment, and in organisms. Several biological fluids contain bacteria and proteins. Both are surface active, that is they prefer to be accommodated at a surface. Usually, the proteins are present in a much higher number than the bacteria and they are much smaller, and, therefore diffuse faster. They easily win the race for the surface and form a film at the surface before the bacteria arrive. The bacteria, that subsequently adhere to the pre-adsorbed protein layer, may proliferate and form a “biofilm” (2). Biofilms are micro-ecosystems in which different strains and species of microorganisms efficiently cooperate in order to protect themselves against environmental stress and to facilitate nutrient uptake (3). In the human body many different biofilms exist. Some of them are essential for the maintenance of health, and offer, for instance, protection against invasion of pathogens (4), whereas other biofilms are detrimental to the body. An example of adverse biofilms is oral biofilm on tooth surfaces. Bacteria in the oral cavity feed on food remnants, notably sugars and other carbohydrates, thereby producing acids that cause dental caries, i.e., the decay of tooth enamel and dentine. Streptococci play a major role in the initiation of dental caries. The interaction between adhering bacteria and the pre-adsorbed salivary conditioning film includes generic, non-specific forces, such as electrostatic, hydrophobic and steric forces. Superimposed on these, specific interactions may play a major role. Specific interactions are usually between a ligand and a receptor and may be referred to as “biological recognition”. Both the non-specific and specific interactions originate from the same fundamental forces, but in specific interactions there is a synergy of different types of forces operating highly directionally in a confined space, yielding strong attraction between the interacting species. Several bacteria have proteinaceous structures on their surface, so-called “adhesins”, that promote their adhesion through biological recognition of saccharides or protein receptors. Bacterial adhesins usually appear in close association with surface appendages. Many proteins have been investigated for their role in adhesion of bacteria to a substratum surface (5–9). The proteins may influence bacterial adhesion because of their adsorption to the substratum surface as well as because of their interaction with the surface of the bacteria. Adhesion of bacteria onto a layer of adsorbed proteins is believed to be important in the pathogenesis of (prosthetic) infections (10, 11). For instance, the Antigen I/II (Ag I/II) family proteins at streptococcal surfaces are involved in specific interactions with salivary proteins (12, 13). The Ag I/II proteins are extended fibrillar structures having a length of about 50 nm (14). They are covalently attached to the bacterial cell wall via their C-terminal end, see Figure 1. 356 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 1. Model of the structure of the Antigen I/II family of proteins, an adhesin at the surface of Streptococcus mutans, that interacts with salivary proteins. Adapted from reference (14).

When extending normally to the cell surface, the projected surface area of AgI/II is estimated to be about 100 nm2. More specifically, Streptococcus mutans is a commensal organism in the human oral cavity and, as a virulent cariogenic species, is often associated with dental caries. It is therefore relevant to investigate the mechanism by which S. mutans adheres to dental surfaces. In vivo, teeth are coated with an organic layer largely composed of adsorbed salivary proteins (15, 16), referred to as “acquired pellicle” or, more generally, the adsorbed salivary conditioning film. Hence, adhesion of oral bacteria to these surfaces is governed by the interaction between the bacteria and constituents of the salivary conditioning film (17). Since Ag I/II proteins at the surface of S. mutans interact specifically with different salivary proteins, it is interesting to investigate the role of Ag I/II in the adhesion of S. mutans to salivary conditioning films. We selected strains of S. mutans with and without Ag I/II at its surface, respectively, that were exposed to salivary proteins. The interaction of the two strains with these proteins were compared using different experimental approaches (18, 19), that is, strength of adhesion by determining the adhesive force using atomic force microscopy (AFM) (20–22), bacterial adhesion to a salivary conditioning film monitored in a parallel plate flow chamber (PPFC), amount of salivary proteins adsorbing to bacterial cell surfaces by determining adsorption isotherms, and enthalpy of saliva-bacterium interaction by isothermal 357 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

titration calorimetry (ITC) (19, 23). Thus, a better understanding of the interaction between S. mutans and salivary proteins has been obtained. It appears to be the outcome of a complex interplay of physical-chemical forces, involving specific and non-specific interactions.


Materials and Methods Culturing and Harvesting Conditions S. mutans LT11 (having Ag I/II at its surface) and the isogenic mutant S. mutans IB03987 (without Ag I/II) were used in this study. The bacterial cells were maintained at ˗80 °C in brain-heart infusion broth (BHI; OXOID, Basingstoke, UK) containing 7% dimethyl sulfoxide (Merck, Darmstadt, Germany). For culturing, S. mutans LT11 was plated onto BHI agar and S. mutans IB03987 onto BHI agar supplemented with 5 μg ml-1 kanamycine monosulfate (Sigma-Aldrich, Steinheim, Germany) and incubated overnight at 37 °C in 5% CO2. Next, bacterial colonies were precultured in 10 ml BHI batch culture overnight. This preculture was used to inoculate a main culture of 200 ml BHI broth, which was allowed to grow overnight. Bacteria were harvested by centrifugation at 6500 g for 5 min at 10 °C and washed twice with demineralized water. Bacterial aggregates were dissociated by mild sonication on ice for 3 x 10 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., Danbury, Ct, USA). Sonication was done intermittently while cooling on ice. This procedure was found to prevent cell lysis of both strains. Finally, the bacteria, having a radius of about 500 nm, were resuspended in phosphate buffer (composition, see below) pH 6.8 or pH 5.8 to concentrations of 5 x 109, 5 x 108, and 5 x 107 cells per ml, as determined in a Bürker-Türk counting chamber. Bacteria were used directly after harvesting.

Saliva Human whole saliva from 20 healthy volunteers was collected into ice-chilled beakers after stimulation by chewing Parafilm. Volunteers gave their consent to saliva donation, in agreement with the Ethics Committee at UMCG (approval no. M09.069104). After pooling and two times centrifugation at 10,000 g for 5 min at 10 °C, phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM to inhibit protease activity. Then the solution was centrifuged again at 10,000 g for 5 min at 10 °C, dialyzed overnight against demineralized water at 4 °C and freeze-dried for storage. Prior to experiments the lyophilized saliva was dissolved in phosphate buffer (composition, see below) pH 6.8 or pH 5.8 to a concentration of 6 mg ml-1. This solution was centrifuged at 10,000 g for 5 min at 10 °C and the supernatant was used in the experiments. The protein content in the supernatant was 1.4 mg ml-1, according to the Bio-Rad protein assay (Bio-Rad Laboratories, San Francisco, CA, USA), which is comparable to the protein concentration in human whole saliva. 358 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Buffer Solution Phosphate buffer containing 2 mM potassium phosphate (K3PO4), 50 mM potassium chloride (KCl) and 1 mM calcium chloride (CaCl2) was used throughout this study. The pH was adjusted to 6.8 or 5.8 by adding hydrochloric acid (HCl).


Strength of Adhesion As Determined by AFM Bacteria were immobilized in a dense layer on an isopore polycarbonate membrane (24). AFM cantilever tips (DNP from Veeco, Woodbury, USA) were mounted in a micromanipulator under microscopic observation to allow only the tip of the cantilever to be coated. A droplet of saliva was placed on a glass slide and the tip of the cantilever was submersed in the droplet for 30 min. The bacterial layer and the protein-coated tip were prepared shortly before measurements. AFM was performed at room temperature in buffer using a Dimension 3100 system (Nanoscope III Digital Instruments, Woodbury, USA). An array of 32 x 32 force-distance curves (approach and retract) at scan rates of 2 Hz were collected over the entire field of view (2 μm x 2 μm). For the conversion of cantilever deflection into force a spring constant of 0.06 N m-1 for the saliva-coated tips was used, determined by the Cleveland method (21). A typical force-distance curve for a probe adhering to a surface is shown in Figure 2. Approach curves were fitted to an exponential function, where the interaction force F is described as

in which F0 is the repulsive force at zero separation between the interacting species, d the separation distance and Λ the decay length of F(d). The strength of adhesion is probed when the saliva-coated tip is retracted from the bacterial surface. The force of adhesion, Fadh, is defined as the minimum in the force-distance curve. To ensure that saliva-coated tips were not damaged during the measurements the force distance curve at 0 s of clean glass were determined before and after scanning the bacterial cell surface. Whenever the 0 s force on the clean glass surface before and after scanning had a difference of ≥ 0.2 nN a new tip was made. Between 200-300 force-distance curves, using bacteria from 5 different cultures, were determined and the distributions of F0, Λ, and Fadh were plotted in histograms from which median values are derived. Bacterial Adhesion Monitored in a PPFC The PPFC and image analysis system have been described in detail in reference (25). Briefly, glass slides (76 mm x 26 mm) were sonicated for 3 min in a surfactant solution (2% RBS 35 detergent in water; Omniclean), rinsed thoroughly with tap water, methanol, tap water, and demineralized water. The bottom plate was coated with a salivary film by immersing the plate overnight at room temperature in the saliva preparation of the desired pH. XPS analysis indicated 359 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


proteinaceous composition of the coating. Prior to each experiment the flow chamber and all tubings were filled with buffer solution, taking care of removing all air bubbles from the system. Next, a bacterial suspension of 5 x 108 cells ml-1 was allowed to flow through the system. Bacterial adhesion to the bottom plate of the flow chamber was monitored in real time, using a phase-contrast microscope (Olympus BH-2) coupled to a CCD-MXR camera (High Technology, Eindhoven, The Netherlands) equipped with 40x ultra-long-working-distance lens (Olympus ULWD-CD plan 40PL). The camera was coupled to an image analyzer (TEA, Difa, Breda, The Netherlands). The flow rate of the bacterial suspension was set at 1.4 ml per min by adjusting the hydrostatic pressure yielding a shear rate of 15 s-1, which for a bacterium with radius 500 nm implies a shear force of about 2.5 x 10-5 nN. Images were taken every 1-2 min during the first 30 min, and thereafter with 10 to 30 min intervals over a period of up to 4 h, where after the flow was stopped. Then, an air bubble was passed through the flow chamber, exerting a shear force of about 10 nN, and the percentage of removed bacteria recorded, giving a semi-quantitative indication of the strength by which the bacteria adhere to the salivary protein layer.

Figure 2. Scheme of a force-distance curve in Atomic Force Microscopy. (1) no interaction force is detected at large separation between the cantilever tip (or probe) and the surface; (2) a repulsive force (——) is detected on approaching the surface and (3) an attractive force (- - - ) as the cantilever is retracted from the surface.

Each image (512 x 512 pixels, with 8-bit resolution) was obtained after summation of 15 consecutive images, taken with 1 s time intervals, to improve the signal-to-noise ratio and to eliminate moving bacteria from the analysis. The surface area grabbed by an image was 0.017 mm2. All experiments were 360 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

performed in 5-fold using separate bacterial cultures. In a parallel experiment, using the buffer solution without bacteria, it was confirmed that the flow of 1.4 ml per min did not remove the protein from the saliva-coated glass, as probed by XPS.


Enthalpy of Saliva-Bacterium Interaction Measured by Isothermal Titration Calorimetry The interaction between salivary components and the bacterial cell surface was probed in a twin-type isothermal microcalorimeter TAM 2277 (Thermometric, Jarfalla, Sweden). In a twin-type calorimeter the heat flowing into or from a reaction ampoule relative to a reference ampoule is channeled through thermocouples (Peltier elements) to a heat sink. The Peltier elements convert the heat into an electrical power, P, signal and, hence, integration of the output signal P(t) equals the heat exchange (26). At constant pressure, p, the heat exchange q equals the enthalpy H effect of the process in the reaction ampoule relative to that in the reference ampoule

The calorimeter contains four identical ampoules (2.2 ml), three of which were filled with 1.5 ml bacterial suspension (5 x 107, 5 x 108, or 5 x 109 bacteria ml-1, respectively) and one, the reference cell, with adhesion buffer. All solutions were stirred with a special home-made two-blades stirrer causing minimum heat effects. The ampoules were lowered gradually in the calorimeter, which was set at 25 °C and left in the measuring position to reach thermal equilibrium. The calorimeter was placed in a room of constant temperature (20 ± 0.1 °C) allowing a baseline stability of ±0.1 μW over 24 h. After reaching a stable baseline, saliva was injected in the ampoules in four consecutive steps of 60 μl with time intervals of 40 minutes, at a controlled rate of 2 μl s-1. All experiments were performed in five-fold. Adsorption Isotherms for Salivary Proteins at the Bacterial Cell Wall In parallel experiments, outside the calorimeter, but under otherwise identical conditions, protein adsorption to the bacterial cell surface was determined. After each injection of salivary proteins solution to the bacterial suspension and allowing for 40 min incubation time the suspension was centrifuged at 10,000 g for 5 min and the protein content in the supernatant was determined by spectrophotometry at 280 nm. The amount of protein adsorbed was derived from mass balance, i.e., the difference between the amounts of protein injected and in the supernatant after adsorption. Titration of buffer solution into bacterial suspension was taken as reference. 361 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.

Statistical Analysis Initial deposition rates and adhesion numbers after 4 h, as determined in PPFC experiments, were subjected to a Student’s t-test to determine significant differences. The AFM data were analyzed using the Statistical Package for the Social Sciences (Version 11.0, SPSS, Chicago, Ill, USA).


Results and Discussion The median values of the repulsive force at contact F0, the decay length Λ of the repulsive force upon approach, and the adhesion force Fadh, all derived from the AFM measurements, are summarized in Table 1. The repulsive force to allow contact between the streptococcal surface and the saliva coated tip is not significantly different between LT11 and IB03987. It suggests that Ag I/II does not play a role in the deposition stage of the bacterium-surface interaction. At pH 5.8 and pH 6.8 the bacteria are negatively charged. In the same buffer as used in our study the zeta-potential of LT11 is ˗15 mV at pH 6.8 and ˗20 mV at pH 5.8 and for IB03987 it is ˗29 mV at pH 6.8 and ˗27 mV at pH 5.8 (27). The salivary conditioning film is negatively charged as well (28, 29). Values for the zeta-potential of a salivary film at enamel and dentin surfaces were reported to be -17 mV and -13 mV, respectively, in the buffer of pH 6.8 (30). At pH 5.8 the charge (and potential) is expected to be somewhat less negative. Hence, the bacterial strain and the salivary coating repel each other electrostatically, the more so the more negative the zeta-potentials of the interacting species are. In line with this, F0 should be much larger for IB03987 than for LT11 at pH 6.8 as well as, but to a lesser extent, at pH 5.8. However, since at both pHs F0 values are more or less the same for the two strains, it is concluded that F0 is not dominated by electrostatic interaction. The same conclusion is reached when comparing F0 values for IB03987 at pH 5.8 and 6.8. Furthermore, according to the Gouy-Chapman model (31), the decay length of the repulsive force between charged species that interact in a buffer of about 0.05 M ionic strength (as in our experiments) is in the range of a few nm, that is, much shorter than the decay lengths derived from the AFM data. Most likely, the repulsive force manifested in the approach curve involves a long-range steric component (32). Upon retraction, attractive adhesion forces may be detected. Figure 3 shows histograms of Fadh for each strain at pH 6.8 and pH 5.8. The fluctuations in the distributions of Fadh is assigned to variations in contact between the salivary protein-coated cantilever tip and heterogeneously distributed surface characteristics of a single bacterial cell. The median adhesion forces are stronger for LT11 than for IB03987, especially at pH 6.8; see Table 1. The median adhesion force for the Ag I/II lacking IB03987 strain is below the detectable limit, both at pH 6.8 and pH 5.8. For the Ag I/II containing LT11 strain Fadh depends strongly on pH, i.e., 0.4 nN at pH 6.8 and 0.1 nN at pH 5.8. The difference in adhesion strength between the two strains points to the involvement of Ag I/II in the interaction with the saliva coating and the influence of pH on Fadh for LT11 suggests participation of ionizable groups (having pK value(s) between 5.8 and 6.8). Furthermore, the observation of an attractive adhesion force only after the 362 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


bacterium has made contact with the salivary coating implies that the adhesive bond is established at close separation in a confined space, which is typical for specific interaction. This, together with the pH-dependence, suggests pairing between oppositely charged groups (in a non-polar environment). In ref. (33) a collection of several single-bond recognition forces is presented revealing that the strength of such a specific bond typically ranges between 0.05 and 0.25 nN. Assuming that the strength of specific bonding between Ag I/II and salivary proteins is in the same range, a force of 0.4 nN (as measured for LT11 at pH 6.8) involves 2-8 Ag I/II molecules. Taking a surface area of 100 nm2 per Ag I/II molecule (see Figure 1) and a contact area of 2000 nm2 between the saliva-coated AFM tip and the bacterial surface the degree of coverage of a LT11 cell by Ag I/II is calculated to be between 10% and 40 %.

Table 1. Median values for the repulsive force at contact F0, the decay length of the repulsive force upon approach Λ, and the adhesive force upon retraction Fadh, for the interaction between a saliva-coated AFM tip and the surface of S. mutans LT11 and IB03987* pH 6.8

*

pH 5.8

LT11

1B03987

LT11

1B03987

F0 (nN)

3.1

3.0

4.6

4.7

Λ (nm)

21

19

23

37

Fadh (nN)

‑0.4

‑0.1

‑0.0

‑0.0

Data taken from reference (18).

The adhesion kinetics of S. mutans LT11 and IB03987 to salivary protein coating at pH 6.8 and pH 5.8, as monitored in a PPFC, are presented in Figure 4. For the different systems the rate of deposition was more or less constant for the first 90 min, except in the case of IB03987 at pH 6.8 where the rate levels off already after about 30 min, reaching a stationary value that is much lower than for the other cases. The observed characteristic features, i.e., the initial deposition rates and the adhesion saturation values, are summarized in Table 2. The deposition rate of bacterial cells in a PPFC, where the flow of the suspension is laminar, is determined by convective diffusion and gravitational sedimentation (34, 35). In systems where specific interactions were not active, deposition rates were found to increase with decreasing repulsive force at contact F0 (36). In the present study this trend is observed as well, not only for IB03987, but also for LT11. It is another indication that specific interaction between Ag I/II and salivary proteins occurs after the bacterium and the saliva-coated surface have been in physical contact. This conclusion is further corroborated by the finding that, for both pH values, the initial rates of deposition do not differ between the two strains. It is further interesting to note that for a given pH (in particular pH 6.8) the ζ-potentials of LT11 and IB03987 are markedly different which is 363 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


not reflected in the initial deposition rate. Hence, the kinetics of deposition on the negatively charged salivary protein film are not governed by electrostatic interactions, as was also concluded from comparison of F0-values (obtained by AFM) between the two strains and at both pHs.

Figure 3. Distribution of the adhesion force Fadh between a saliva-coated AFM tip and surfaces of S. mutans LT11 and IB03987. Each histogram is based on 200-300 force-distance curves, equally divided over five different bacteria. Redrawn from reference (18).

Figure 4. Kinetics of adhesion of S. mutans LT11 and IB03987 to salivary coatings, determined in a parallel plate flow chamber. Redrawn from reference (18). 364 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


In all cases the number of bacteria adhering after 4 h represent adhesion saturation (under the prevailing conditions). In the absence of specific interaction with the salivary proteins, that is for IB03987, adhesion saturation is much higher at pH 5.8 than at pH 6.8. Although the initial deposition rate was found not to be significantly influenced by electrostatic interactions, adhesion saturation, that is the steady-state density of adhering cells per unit area of the substrate surface, may well be affected by electrostatic interaction. The ζ-potential of IB03987 is reported to be about the same at pH 5.8 and pH 6.8 (˗ 27 mV and ˗ 29 mV, respectively) (27), and assuming that the charge density of the salivary protein coating is more negative at higher pH, adhesion is electrostatically less favorable at higher pH. However, it seems unlikely that electrostatic effects are primarily responsible for the large difference in adhesion saturation of IB03987 at the two pH values. For LT11 the influence of pH on adhesion saturation has largely disappeared or, otherwise stated, at pH 6.8 adhesion saturation is much higher for LT11 than for IB03987. This may be a manifestation of favorable specific bonds formed between Ag I/II moieties at the LT11 cell surface and the salivary proteins. The slightly higher adhesion saturation for LT11 as compared to IB03987, at pH 5.8, then suggests that, at this pH value, AgI/II is much less involved in specific interaction.

Table 2. Interaction between S. mutans LT11 and IB03987 and a salivary coating in a parallel plate flow chamber. Initial deposition rate, number of adhering bacteria per unit surface area after 4 h, and percentage of bacteria detached by a passing air bubble. Experiments were done in five-fold with separately prepared saliva-coated glass plates and different bacterial cultures* pH 6.8

*

pH 5.8

LT11

1B03987

LT11

1B03987

initial deposition rate (cm2 s‑1)

1679 ± 165

1441 ± 119

1315 ± 28

1258 ± 169

adhesion after 4 h (106 cm‑2)

9.6 ± 2.3

2.5 ± 0.7

12.7 ± 1.1

10.5 ± 2.1

detachment by passing air bubble (%)

55 ± 9

76 ± 2

73 ± 3

76 ± 5

Data taken from reference (18).

Finally, the percentages of bacterial cells that are detached by a passing air bubble, given in Table 2, indicate strong adhesion of LT11 at pH 6.8, in line with the values of Fadh, derived from AFM.

365 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


In Figure 5 adsorption isotherms are presented, where the amounts of salivary proteins adsorbed per unit area bacterial surface are plotted as a function of the protein concentration in solution after adsorption. In all cases adsorption appeared to be irreversible towards variation of dissolved protein concentration. This is a common feature of protein adsorption (37). However, exchange of protein between the adsorbed and the dissolved state may well be possible (38). Since the adsorption isotherms reflect partitioning of the proteins between the surface (mg m-2) and the solution (mg ml-1) they are insensitive to the total surface area, i.e., the number of bacteria, in the system.

Figure 5. Adsorption isotherms for salivary proteins on the surfaces of S. mutans LT11 and IB03987, obtained after consecutive injections of 60 μl salivary protein solution (1.4 mg ml-1) into 1.5 ml bacterial suspension containing (●) 5 x 10-9, (○) 5 x 10-8, (▼) 5 x 10-7 bacteria per ml. Redrawn from reference (19). Clearly, because the adsorbed amount is derived from the difference in solution concentration before and after adsorption, the experimental error is larger when that difference is smaller, that is when a smaller number of bacterial cells is supplied. The adsorption isotherms show that the affinity of salivary proteins to adsorb at the S. mutans surface is much higher for the LT11 strain, demonstrating the role of Ag I/II in the binding process. The lower protein adsorption at the LT11 surface at pH 5.8 as compared to pH 6.8 points to decreased specific binding via Ag I/II, in line with the AFM and PPFC results. Protein adsorption at full coverage of the substrate surface typically is in the range of a few mg m-2 (the exact value depends on the size, shape and orientation of the protein molecule) (37, 39); hence, it appears that adsorption of salivary proteins at the S. mutans surface is compatible with (sub-)monolayer coverage. At constant pressure and temperature the affinity of the protein-bacterium interaction is given by the change in Gibbs energy, ΔG, of the system resulting from that interaction. The more negative ΔG , the higher the affinity is, or, in other 366 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


words, the stronger the adhesive bond. Because the adsorption isotherms do not represent reversibility with respect to protein concentration in solution, they do not allow for deduction of ΔG (40). The value of ΔG is composed of a change in enthalpy, ΔH, and in entropy ΔS, according to

where T is the temperature in Kelvin. For irreversible processes ΔS is not experimentally accessible either, but ΔH may be determined calorimetrically as the heat exchange between the system and its environment, as given by equation (2). At constant volume, which is practically the case when no gaseous components are involved in the interaction, the enthalpy change equals the change in energy. Enthalpy changes due to the adsorption of salivary proteins to the surface of S. mutans cells are, for each injection step, presented in Figure 6. Enthalpy effects arising from metabolic activity is minimal because all experiments are carried out in the absence of nutrients. Note that the adsorbed amount after each injection step can be read off from the adsorption isotherms in Figure 5. AFM data and adsorption isotherms indicated that the non-specific interaction of salivary proteins with the Ag I/II-lacking surface of the IB03987 is relatively weak. The calorimetric data in the lower panels of Figure 6 reveal that this interaction is essentially a-thermal, i.e., ΔH ≈ 0. It implies that the driving force for this non-specific interaction is entropy gain, ΔS > 0. Entropy-driven adsorption has often been reported for proteins. Adsorption of salivary proteins to the Ag I/II-containing LT11 cells is strongly exothermic, i.e., ΔH < 0 (Figure 6, upper panels). Apparently, the specific interaction between Ag I/II and salivary proteins is energetically favorable. The difference in ΔH between pH 6.8 and pH 5.8 indicates suppression of Ag I/II involvement at the lower pH, just as is concluded from adsorption isotherms, AFM and PPFC experiments. It is further worth mentioning that a 100-fold increase in bacterial concentration (= 100-fold increase in available surface area) yields only a 4-5 fold increase in adsorption enthalpy. It means that at higher supply of sorbent surface area the enthalpy change per bacterial cell decreases drastically. In this context, it is instructive to plot ΔH per bacterium as a function of the amount of protein adsorbed per bacterial cell (taken from the isotherms in Figure 5). The result is presented in Figure 7, showing that only for LT11 at pH 6.8, and only in case of not too high bacterial concentration, a significant enthalpy effect per bacterium can be detected. Thus, for LT11, pH 6.8, it is established that the enthalpy per bacterium increases, i.e., becomes more negative, with increasing surface coverage up to a value beyond which further adsorption proceeds a-thermally. That value may mark full occupation of Ag I/II binding sites at the bacterial surface. This occurs at about 4 x 10-12 mg protein per bacterium, which, for a spherical bacterium having a radius of 500 nm, corresponds to 1.25 mg m-2. Comparison with the semi-plateau value of the adsorption isotherm of LT11 at pH 6.8 (Figure 5), it is inferred that saturation of the bacterial surface with salivary proteins is dominated by Ag I/II mediated specific interaction, but leaving some space on the surface for non-specific adsorption. 367 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


Figure 6. Enthalpy of adsorption of salivary proteins to the surface of S. mutans LT11 and IB03987 upon consecutive injections into bacterial suspensions. Conditions and symbols as in Figure 5. Redrawn from reference (19).

Figure 7. Enthalpy of adsorption of salivary proteins to the surface of S. mutans LT11 and IB03987 as a function of the amount of protein adsorbed at the bacterial surface. Symbols as in Figure 5. 368 In Proteins at Interfaces III State of the Art 2012; Horbett, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2012.


The smaller enthalpy change per bacterium at higher bacterial concentration in a suspension containing a given amount of saliva may be due to the presence of a larger number of Ag I/II binding sites. Douglas and Russell (41) have identified various salivary proteins that have a preference to bind to the surface of S. mutans. They probably bind with different enthalpy effects. In case of the lowest bacterial concentration (5 x 107 cells ml-1) the number of Ag I/II moieties is limited and, therefore, they all become saturated with the enthalpically most favorably binding proteins. The highest bacterial concentration (5 x 109 cells ml-1) offers 100x more binding sites to the same amount of salivary proteins. Then, a much smaller fraction of the Ag I/II binding sites interacts with the strongest binding protein, another fraction with the second strongest binding protein, etcetera. The result is a much lower enthalpy effect per bacterium.

Conclusions Using two Streptococcus mutans strains, one with the adhesin Antigen I/II at its surface and its isogenic mutant that does not contain the adhesin, we were able to distinguish between generic and specific contributions to their interaction with salivary proteins. Results obtained from a variety of experiments, i.e., kinetics of deposition of the bacteria on a saliva-coated surface, binding isotherms and enthalpy of adsorption of salivary proteins at the bacterial cell walls, reveal a major contribution of specific interaction between the Antigen I/II containing S. mutans strain and salivary proteins. This conclusion is corroborated by a dominant contribution from specific interaction to the adhesive bond strength, as measured by retracting a saliva-coated AFM tip from the surface of the bacterial cells. It was deduced that the generic interaction between the bacterium and the surface is longranged, whereas the specific interaction, involving Antigen I/II, is short-ranged, which is typical for biological recognition. This short-range interaction appears to be strongly pH-dependent suggesting the involvement of ion pairing, which, in a confined space that is more or less shielded from the aqueous environment, leads to the observed strong attractive force causing a highly exothermic effect. This study demonstrates how a multi-sided experimental approach provides insight in the mechanism of S. mutans-salivary proteins interaction. Following a similar approach may as well be successful in investigating other biological recognition processes.

Acknowledgments We acknowledge ZON-MW for financial support (Grant 9110)

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Specific and Nonspecific Interactions between Salivary Proteins and

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