Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.


Introduction
Streptococcus mutans is one of the most important etiologic agents of dental caries [1,2]. It efficiently colonizes the oral cavity because of its ability to form biofilms on dental surfaces. When S. mutans is grown in the presence of sucrose, it produces glucans and fructans that contribute to the formation of a dense, adherent biofilm that allows acids to accumulate on the tooth surface, resulting in enamel demineralization [3]. S. mutans can also form biofilms by a sucrose-independent mechanism [4].
Under certain circumstances (dental procedures, oral infections, dental hygiene, and eating), S. mutans can gain access to the bloodstream, thus causing a transient bacteremia [5]. Nakano et al. recently reported that S. mutans was the oral bacterial species most frequently detected in cardiovascular specimens, suggesting that it enters the bloodstream more readily than other oral bacteria [6]. While, in most cases, the bacteremia has no serious consequences, S. mutans can adapt to this environment, resist to the immune defense, colonize the heart, and cause infective endocarditis, a severe and often fatal systemic disease [7]. Individuals with congenital cardiac malformations, with prosthetic aortic valves or having a compromised immune system, are more susceptible to develop infective endocarditis [7]. S. mutans is commonly isolated in cases of infective endocarditis, which is a typical biofilm-associated infection [8]. Bacteria colonizing the host in biofilms are more resistant to the host immune defense and to killing by antibiotics in comparison with planktonic bacteria [9].
The ability of S. mutans to colonize the cardiac valves and form biofilms is critical for its capacity to cause infective endocarditis. Recently, Jung et al. [10] brought in vitro and in vivo evidence indicating that host factors can modulate biofilm formation in S. mutans. More specifically, they showed that interactions between platelets and S. mutans play a key role in biofilm formation [10]. It is likely that additional host factors also contribute to promote the formation of biofilm by S. mutans. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced and the impact of fibrinogen on the resistance to penicillin of S. mutans were also evaluated.

Effects of Plasma and Various Plasma Proteins on Biofilm
Formation. The ability of S. mutans to form biofilms when grown in the presence of bovine plasma (Sigma-Aldrich Canada Co., Oakville, ON, Canada; 100%, 50%, 25%, and 12.5% in 10 mM phosphate-buffered saline (PBS, pH 7.2)) was tested in 96-well polystyrene tissue culture plates. The effect of various plasma proteins (5 mg mL −1 ), including human fibrinogen (EMD Millipore, Billerica, MA, USA), humanglobulin (Sigma-Aldrich Canada Co.), bovine serum albumin (BSA) (Fisher Scientific Company, Ottawa, ON, Canada), and human transferrin (Sigma-Aldrich Canada Co.), on biofilm formation by S. mutans was evaluated in a similar fashion. The effect of twofold serial dilutions of human fibrinogen (5 to 0.039 mg mL −1 ) in THB-HK medium was also tested. Lastly, biofilm formation in THB-HK medium with and without fibrinogen (5 mg mL −1 ) and supplemented with twofold serial dilutions of sucrose (1% to 0.0156%) was tested. An overnight culture of S. mutans was diluted in fresh THB-HK medium to get an optical density at 655 nm (OD 655 ) of 0.2 (10 8 colony forming units (cfu) mL −1 ). Samples (100 L) were added to the wells of a 96-well polystyrene tissue culture plate containing 100 L of THB-HK medium ± the protein described above. After a 48 h incubation at 37 ∘ C, the OD 655 was measured to estimate bacterial growth. Culture medium and free-floating bacteria were then removed by aspiration, the wells were washed twice with PBS, and the biofilms were stained with 0.05% crystal violet dye (100 L) for 10 min. The wells were washed twice with PBS to remove unbound crystal violet dye and were dried for 2 h at 37 ∘ C. After adding 100 L of 95% (v/v) ethanol to each well, the plate was shaken for 10 min to release the stain from the biofilms, and the absorbance at 550 nm (A 550 ) was determined to quantify biofilm formation. Uninoculated wells containing culture medium served as controls.

Scanning Electron Microscopy.
The structural architecture of S. mutans biofilms formed in the presence of fibrinogen (0.156 mg mL −1 ), plasma (50%), or sucrose (0.25%) was observed by scanning electron microscopy. Briefly, S. mutans suspended at a final OD 655 of 0.1 in plasma or THB-HK containing one of the test compounds was added (2 mL) to the wells of a 6-well polystyrene tissue culture plate, each well containing a 13 mm diameter plastic coverslip (Nunc, Kastrup, Denmark). The culture plate was incubated at 37 ∘ C for 48 h. The culture medium and free-floating bacteria were then removed by aspiration. The biofilm-coated coverslips were incubated overnight at 4 ∘ C in fixation buffer (4% paraformaldehyde, 2.5% glutaraldehyde, and 2 mM CaCl 2 in 0.2 M cacodylate buffer, pH 7.2), washed twice with 0.1 M cacodylate buffer (pH 7.0), and postfixed for 90 min at room temperature in 1% osmic acid containing 2 mM potassium ferrocyanide and 6% sucrose in 0.1 M cacodylate buffer (pH 7.0). The biofilms on the coverslips were dehydrated using a graded series of ethanol (50%, 70%, 95%, and 100%), critical point dried, gold sputtered, and examined using an electron microscope (JEOL JSM6360LV; JEOL, Tokyo, Japan) operating at 15 kV.

Determination of Minimal Inhibitory and Minimal
Bactericidal Concentrations of Penicillin. The minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) of penicillin for S. mutans grown in THB-HK were determined using a microbroth dilution method. The wells of a 96-well microplate, each well containing 100 L of serially diluted penicillin (penicillin G; 20-0.039 g mL −1 in THB-HK), were inoculated with 100 L of an overnight culture of S. mutans diluted in fresh culture broth to obtain an OD 655 of 0.2 (10 8 CFU mL −1 ). The MIC was the lowest concentration of antibiotic for which no significant increase in OD 655 was noted after a 24 h incubation at 37 ∘ C. To determine the MBC, 10 L of culture was collected from wells with no apparent growth and spread on THB-HK agar plates. The MBC was the lowest concentration of antibiotic at which no colonies grew on the plates after 48 h of incubation at 37 ∘ C. The MIC and MBC for biofilm-grown S. mutans were determined using a microplate containing a 24 h preformed fibrinogeninduced biofilm in each well. The culture supernatants were aspirated, and 200 L of twofold serial dilutions of penicillin in fresh culture broth was added to the wells. The plate was incubated at 37 ∘ C for 24 h. The biofilm-grown bacteria were then suspended by scraping the bottom of the wells with a 200 L pipet tip and then pumping the solution up and down until a homogenous suspension was obtained. Growth was then estimated by recording the OD 655 . The MIC was the lowest concentrations of penicillin for which no significant increase in OD 655 was noted. The MBC of the biofilm-grown cells was determined by spreading 10 L of the resuspended biofilm on a THB-HK agar plate. The MBC was the lowest concentration of antibiotic for which no colonies grew on the agar medium after 48 h of incubation.

Effect of Fibrinogen on the Adherence of S. mutans to
Endothelial Cells. The effect of fibrinogen on the adherence of fluorescein isothiocyanate (FITC)-labeled S. mutans to human endothelial cells was evaluated. A 10 mL aliquot of a 24 h culture of S. mutans was centrifuged (7000 ×g for 10 min), and the pellet was suspended in 12 mL of 0.5 M NaHCO 3 (pH 8) containing 0.03 mg mL −1 FITC. The bacterial suspension was incubated in the dark at 37 ∘ C for 30 min with constant shaking. The bacteria were then washed three times by centrifugation (7000 ×g for 5 min) and were suspended in the original volume of PBS. Immortalized human brain microvascular endothelial cells kindly provided by Dr. Marcelo Gottschalk (Université de Montréal, QC, Canada) were grown in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated FBS, 10% Nu-serum IV supplement (BD Biosciences, Bedford, MA, USA), and 50 g mL −1 of penicillin-streptomycin, until they reached confluence, as previously described [11]. The cells were harvested by gentle trypsinization with 0.05% trypsin-ethylenediaminetetraacetic acid (Invitrogen, Grand Island, NY, USA) at 37 ∘ C and were suspended in RPMI-1640 medium (without FBS and Nu-serum). Aliquots of cell suspension (100 L, 1.5 × 10 6 cells mL −1 ) were placed in the wells of 96-well black plates (Greiner Bio-One, St. Louis, MO, USA). After a 4 h incubation at 37 ∘ C in a 5% CO 2 atmosphere to allow a confluent monolayer to form, spent medium was aspirated, 100 L of 1% glutaraldehyde was added to the wells, and the plate was incubated at 4 ∘ C overnight. Glutaraldehyde was aspirated, and the wells were washed three times with PBS. Filtered 1% bovine serum albumin (100 L) was added to each well, and the plate was incubated for 30 min at 37 ∘ C. The wells were washed once with PBS, 100 L of fibrinogen (final concentration: 0, 0.01, 0.1, 1, and 10 mg mL −1 ) was added to each well, and the plate was incubated for 30 min. FITClabeled S. mutans cells were then added (100 L) to the wells at a multiplicity of infection (MOI) of 200, and the plate was incubated in the dark for 2 h at 37 ∘ C. Unbound bacteria were removed by aspiration, and the wells were washed three times with PBS. Relative fluorescence units (RFU; excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to the level of bacterial adherence were determined using a microplate reader. Control wells without fibrinogen were used to determine the 100% adherence value. Wells containing only endothelial cells and fibrinogen were also prepared to determine the autofluorescence values related to fibrinogen.

Assay for Fibrinogen Conversion into
Fibrin. The ability of S. mutans (cells and culture supernatant) to convert fibrinogen into fibrin was assayed using a plate assay, as previously described [12]. Briefly, a solution containing 1.2% agarose and 0.4% human fibrinogen was prepared in 100 mM tris-HCl buffer (pH 7.4), and 10 mL was poured into a 100 × 15 mm Petri dish. After solidification, wells (7 mm diameter) were cut in the agarose and were filled with 120 L of S. mutans cell suspension (OD = 1.0) or culture supernatant. Thrombin (0.4 unit) was used as a positive control and tris-HCl (pH 7.4) was used as a negative control. After a 5 h incubation at 37 ∘ C, the Petri dish was examined. The presence of an opaque zone around a well indicated that the fibrinogen had been converted into fibrin.

Assay for Fibrinogen-Binding
Activity. The fibrinogenbinding activity of S. mutans cells was investigated using human fibrinogen-Alexa Fluor conjugate (Invitrogen). Briefly, bacteria from an overnight culture of S. mutans were harvested by centrifugation and were suspended in PBS containing fluorescent fibrinogen (40 g/mL). The mixture was incubated in the dark at room temperature for 60 min. The bacteria were then washed three times with PBS by centrifugation and were suspended in the original volume of PBS. Aliquots of bacterial suspension (100 L) were placed in the wells of 96-well black plates (Greiner Bio-One). RFU (excitation wavelength 495 nm; emission wavelength 525 nm) corresponding to fibrinogen bound to S. mutans were determined using a microplate reader and were compared to a standard curve generated using fluorescent fibrinogen at different concentrations. As a control, S. mutans cells were preincubated with nonfluorescent fibrinogen (0.5, 0.25, and 0.15 mg mL −1 ) for 1 h at room temperature prior to incubating them in the presence of fibrinogen-Alexa Fluor conjugate.

Statistical Analysis.
All the experiments were run in triplicate in two independent experiments, and the means ± standard deviations (SD) were calculated. The statistical analysis was performed using Students t-test.

Results
S. mutans (ATCC 25175) formed a more significant biofilm when grown in bovine plasma than in THB-HK (Figure 1). In undiluted and 12.5% (v/v) plasma, the amount of biofilm increased 7-and 4-fold, respectively, compared to the biofilm formed in THB-HK. To identify the plasma component that induced biofilm formation, S. mutans (ATCC 25175) was grown in THB-HK supplemented with various plasma proteins. Fibrinogen promoted the formation of biofilm, while albumin, transferrin, and -globulin had no significant effect ( Figure 2). Moreover, fibrinogen dose dependently induced biofilm formation by S. mutans. At the highest concentration of fibrinogen tested (5 mg mL −1 ), the amount of biofilm formed was approximately 5-fold higher than that formed in unsupplemented culture medium (Figure 3). Even at the lowest concentration tested (0.039 mg mL −1 ), fibrinogen had a significant effect on biofilm formation. Fibrinogen-induced biofilm formation was not related to a growth-promoting effect since the final OD 655 was the same in all the assays (data not shown). To determine whether the above phenomenon was strain specific, additional strains of S. mutans were tested. As reported in Table 1, the fibrinogen-induced biofilm formation was observed for five out of eight strains of S. mutans. Further analyses were carried out with strain ATCC 25175 (serotype c).
The structural architecture of the S. mutans biofilm formed in the absence and presence of human fibrinogen was examined by scanning electron microscopy. In the absence of  fibrinogen, short chains of S. mutans were observed attached to the polystyrene surface and were rarely bound to each other (Figure 4(a)). However, when the culture medium was supplemented with fibrinogen (0.156 mg mL −1 ), aggregates and microcolonies of S. mutans completely covered the surface of the support (Figure 4(c)). Similar results were observed when S. mutans was grown in the presence of 50% plasma (Figure 4(b)) and 0.25% sucrose (Figure 4(d)). We then tested whether sucrose and fibrinogen in combination had an additive effect on biofilm formation by S. mutans. As expected, sucrose (starting at 0.031%) had a dosedependent effect on the amount of biofilm formed ( Figure 5). However, no additive effect was observed in the presence of both sucrose and fibrinogen. Results are expressed as means ± standard deviations of triplicate assays from two independent experiments. * < 0.05, * * < 0.01, and * * * < 0.001: significantly different from the control (without fibrinogen). The MIC and MBC of penicillin G for S. mutans grown in THB-HK (planktonic form) were 0.0195 and 1.25 g mL −1 , respectively. The MIC and MBC for biofilm-grown S. mutans were also determined using preformed fibrinogen-induced biofilms. In this case, the MIC was the same as that for the planktonic form of S. mutans, while the MBC was 5 g mL −1 .
We then evaluated the capacity of fibrinogen to promote the adherence of S. mutans to endothelial cells. Fibrinogen dose dependently increased bacterial adherence to endothelial cells (Figure 6). At the highest concentration tested (5 mg mL −1 ), fibrinogen increased the adherence of S. mutans to endothelial cells by 75%. However, no significant differences were observed at the lowest concentrations tested (0.005 and 0.05 mg mL −1 ).
The potential mechanisms by which human fibrinogen promotes S. mutans biofilm formation and adherence were then investigated. First, the ability of S. mutans (cells and culture supernatant) to convert fibrinogen into fibrin was assayed using a plate assay. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin, while the positive control (thrombin) produced an opaque zone (data not shown). We then evaluated the capacity of S. mutans to bind fibrinogen to its cell surface, a phenomenon that may result in bridging and thus contributes to biofilm formation. S. mutans cells bound fibrinogen-Alexa Fluor conjugate (Figure 7). Pretreating S. mutans cells with nonfluorescent fibrinogen prior to incubating them with fibrinogen-Alexa Fluor conjugate resulted in a significant inhibition of fluorescence, suggesting that the binding is specific.

Discussion
The ability of bacteria to form biofilms on host surfaces is a crucial virulence factor and protects them against innate host defenses and antimicrobial agents. The formation of biofilms is important in the pathogenesis of several subacute and chronic human bacterial infections, including endocarditis [13]. Bacterial endocarditis is frequently caused by commensal streptococci such as S. mutans that colonizes the cardiac valve in a biofilm composed of bacteria and their extracellular products, as well as host components (platelets, fibrin) [13]. In the present study, we showed that S. mutans biofilm formation on a polystyrene surface is promoted by plasma and that fibrinogen is the component responsible for this effect. Fibrinogen is a 340 kDa glycoprotein in human blood plasma that is involved in blood coagulation through its conversion into fibrin by thrombin [14]. The concentrations of fibrinogen required to induce biofilm formation by S. mutans are relevant to in vivo situations since the normal levels of fibrinogen in plasma are in the range of 1 to 4.5 mg mL −1 . Jung et al. [10] previously reported that platelets are required for S. mutans biofilm formation in plasma, while our study showed that fibrinogen alone is sufficient. This discrepancy may be related to the bacterial strain used since we showed that fibrinogen-induced biofilm formation is not observed for all isolates of S. mutans.
In our study, S. mutans growing in a fibrinogen-induced biofilm was found to be more resistant to the bactericidal activity of penicillin. This enhanced resistance to penicillin was likely related to the stable architecture of the biofilm, which restricted the penetration of the antibiotic. The presence of persister cells (dormant bacteria) may also explain the resistance of S. mutans in biofilms to killing by penicillin, which acts on growing cells.
Fibrinogen also increased the adherence of S. mutans to endothelial cells. Since S. mutans is known to invade human coronary artery endothelial cells [15] and that close interactions between bacteria and host cells are critical in the invasive process, this fibrinogen-induced adhesion may enhance the ability of S. mutans to enter endothelial cells and avoid being eliminated by the immune system. Interestingly, Cheung et al. reported that fibrinogen can act as a bridging molecule to promote the adherence of Staphylococcus aureus to cultured endothelial cells [16].
To identify the mechanism by which fibrinogen promotes biofilm formation by S. mutans, we evaluated the ability of this species to convert fibrinogen into fibrin and to bind fibrinogen to its cell surface. While S. mutans could not mediate the conversion of fibrinogen into fibrin, it possessed a fibrinogen-binding activity. This is in agreement with previous studies reporting that the major surface antigen I/II of S. mutans is involved in the fibrinogen-binding activity [17,18]. This property may allow bacteria to attach to each other through fibrinogen-mediated crossbridging. Fibrinogen binding to streptococci also plays a significant role in enabling them to adhere to host surfaces and in protecting them from the host immune system, notably by preventing opsonophagocytosis [19,20]. In a previous study, we showed that fibrinogen can specifically induce biofilm formation by Streptococcus suis, which is a major causative agent of infective endocarditis in swine [21]. As for S. mutans, S. suis can express fibrinogen surface receptors allowing fibrinogen to act as a bridging molecule.

Conclusions
Our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells. Fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule. This phenomenon may contribute to infective endocarditis.