Saliva-coated Surfaces Research Articles

Prevention of Initial Bacterial Attachment by Osteopontin and Other Bioactive Milk Proteins.

A considerable body of work has studied the involvement of osteopontin (OPN) in human physiology and pathology, but comparably little is known about the interaction of OPN with prokaryotic cells. Recently, bovine milk OPN has been proposed as a therapeutic agent to prevent the build-up of dental biofilms, which are responsible for the development of caries lesions. Bioactive milk proteins are among the most exciting resources for caries control, as they hamper bacterial attachment to teeth without affecting microbial homeostasis in the mouth. The present work investigated the ability of OPN to prevent the adhesion of three dental biofilm-forming bacteria to saliva-coated surfaces under shear-controlled flow conditions in comparison with the major milk proteins α-lactalbumin, β-lactoglobulin, αs1-casein, β-casein and κ-casein, as well as crude milk protein. OPN was the most effective single protein to reduce the adhesion of Actinomyces naeslundii, Lactobacillus paracasei subsp. paracasei and Streptococcus mitis. β-casein and crude milk protein also had a pronounced effect on all three species, which suggests binding to different microbial surface structures rather than the blocking of a specific bacterial adhesin. Bioactive milk proteins show potential to delay harmful biofilm formation on teeth and hence the onset of biofilm-related oral disease.

Influence of environmental factors on the two-species biofilm formed by Streptococcus oligofermentans and Streptococcus mutans

Objective: To study the influence of environmental factors on the two-species biofilm formed by the combinations of Streptococcus oligofermentans (So) with Streptococcus mutans (Sm) and Streptococcus sanguinis (Ss) with Sm so as to evaluate the role of So in maintaining the microecological balance of the oral cavity. Methods: Single-and two-species biofilms were grown on saliva-coated surfaces (glass tube and 96-well plate). Colony-counting method and safranin staining method were used to measure the biofilms formed under various oxygen conditions (aerobic and anaerobic), sucrose conditions (0%, 1% and 5% sucrose concentrations) and pH conditions (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0). Results: Comparing the numbers of Sm in two co-cultures under various conditions, Sm counts in So+Sm group [(7.70±2.46)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(9.00±1.13)×10(8) CFU/ml] in aerobic environment (P<0.05). Sm counts in So+Sm group [(2.80±0.52)×10(8) CFU/ml] were also significantly lower than those in the Ss+Sm group [(4.00±1.25)×10(8) CFU/ml] in anaerobic environment (P<0.05). The Sm counts in So+Sm group [(8.90±0.82)×10(8) CFU/ml] were significantly higher than those in Ss+Sm group [(7.50±1.73)×10(8) CFU/ml] in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group [(5.70±2.94)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.30±3.21) ×10(8) CFU/ml] in 1% sucrose environment (P<0.05). The Sm counts in So+Sm group [(6.10±1.71)×10(8) CFU/ml] were also significantly lower than those in Ss+Sm group [(7.40±1.20)×10(8) CFU/ml] in 5% sucrose environment (P<0.05). The Sm counts in So+Sm group [(3.50±1.50)×10(8) CFU/ml] were significantly lower than those in Ss+Sm group [(10.70±2.80)×10(8) CFU/ml] in pH7.0 environment (P<0.05). Comparing the formation of biofilm after 24 h cultivation, the Sm counts in So+Sm group were significantly lower than those in Ss+Sm group both in aerobic and anaerobic environments (P<0.05). The Sm counts in So+Sm group were significantly higher than those in Ss+Sm group in 0% sucrose environment (P<0.05). The Sm counts in So+Sm group were significantly lower than those in Ss+Sm group in 1% and 5% sucrose and pH 7.0 environments (P<0.05). Both So and Ss had no inhibitory effect on Sm in pH5.5 and pH8.0 environments. Conclusions: In the in vitro two-species co-culture systems, So showed stronger inhibitory effects than Ss on Sm and its inhibitory ability might influenced by various environmental factors.

Crystalline anatase-rich titanium can reduce adherence of oral streptococci

Dental implant abutments that emerge through the mucosa are rapidly covered with a salivary protein pellicle to which bacteria bind, initiating biofilm formation. In this study, adherence of early colonizing streptococci, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Streptococcus sanguinis to two saliva-coated anodically oxidized surfaces was compared with that on commercially pure titanium (CpTi). Near edge X-ray absorption (NEXAFS) showed crystalline anatase was more pronounced on the anodically oxidized surfaces than on the CpTi. As revealed by fluorescence microscopy, a four-species mixture, as well as individual bacterial species, exhibited lower adherence after 2 h to the saliva-coated, anatase-rich surfaces than to CpTi. Since wettability did not differ between the saliva-coated surfaces, differences in the concentration and/or configuration of salivary proteins on the anatase-rich surfaces may explain the reduced bacterial binding effect. Anatase-rich surfaces could thus contribute to reduced overall biofilm formation on dental implant abutments through diminished adherence of early colonizers.

Effect of neovestitol–vestitol containing Brazilian red propolis on accumulation of biofilm in vitro and development of dental caries in vivo

The present study examined the influences of the neovestitol–vestitol (NV) containing fraction isolated from Brazilian red propolis on the development of biofilm and expression of virulence factors by Streptococcus mutans using saliva-coated surfaces of hydroxyapatite. In addition, NV was tested in a rodent model of dental caries to assess its potential effectiveness in vivo. Topical applications of NV (800 μg ml−1) significantly impaired the accumulation of biofilms of S. mutans by largely disrupting the synthesis of glucosyltransferase-derived exopolysaccharides and the expression of genes associated with the adaptive stress response, such as copYAZ and sloA. Of even greater impact, NV was as effective as fluoride (positive control) in reducing the development of carious lesions in vivo. NV is a promising natural anti-biofilm agent that targets essential virulence traits in S. mutans, which are associated with the formation of cariogenic biofilm and the subsequent onset of dental caries disease.

Inhibition of initial adhesion of oral bacteria through a lectin fromBauhinia variegataL. var. variegata expressed inEscherichia coli

The aim of the present work was to study the in vitro effect of native and recombinant Bauhinia variegata var. variegata lectins in inhibiting early adhesion of Streptococcus mutans, Streptococcus sanguis and Streptococcus sobrinus to experimentally acquired pellicle. Native lectin from B. variegata (BVL) was purified by affinity chromatography of extract of seeds. The recombinant lectin (rBVL-I) was expressed in E. coli strain BL21 (DE3) from a genomic clone encoding the mature B. variegata lectin gene using the vector pAE-bvlI. Recombinant protein deposited in inclusion bodies was solubilized and subsequently purified by affinity chromatography. The rBVL-I was compared to BVL for agglutination of erythrocytes and initial adherence of oral bacteria on a saliva-coated surface. The results revealed that rBVL-I acts similarly to BVL for agglutination of erythrocytes. Both lectins showed adhesion inhibition effect on Step. sanguis, Step. mutans and Step. sobrinus. We report, for the first time, the inhibition of early adhesion of oral bacteria by a recombinant lectin. Our results support the proposed biotechnological application of lectins in a strategy to reduce development of dental caries by inhibiting the initial adhesion and biofilm formation.

Effect of Osteopontin on the Initial Adhesion of Dental Bacteria

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.

Osteopontin Reduces Biofilm Formation in a Multi-Species Model of Dental Biofilm

BackgroundCombating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.Methodology/Principal FindingsBiofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.Conclusions/SignificanceOPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.

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

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

Streptococcus pyogenes antigen I/II-family polypeptide AspA shows differential ligand-binding properties and mediates biofilm formation.

The streptococcal antigen I/II (AgI/II)-family polypeptides are cell wall-anchored adhesins expressed by most indigenous oral streptococci. Proteins sharing 30–40% overall amino acid sequence similarities with AgI/II-family proteins are also expressed by Streptococcus pyogenes. The S. pyogenes M28_Spy1325 polypeptide (designated AspA) displays an AgI/II primary structure, with alanine-rich (A) and proline-rich (P) repeats flanking a V region that is projected distal from the cell. In this study it is shown that AspA from serotype M28 S. pyogenes, when expressed on surrogate host Lactococcus lactis, confers binding to immobilized salivary agglutinin gp-340. This binding was blocked by antibodies to the AspA-VP region. In contrast, the N-terminal region of AspA was deficient in binding fluid-phase gp-340, and L. lactis cells expressing AspA were not agglutinated by gp-340. Deletion of the aspA gene from two different M28 strains of S. pyogenes abrogated their abilities to form biofilms on saliva-coated surfaces. In each mutant strain, biofilm formation was restored by trans complementation of the aspA deletion. In addition, expression of AspA protein on the surface of L. lactis conferred biofilm-forming ability. Taken collectively, the results provide evidence that AspA is a biofilm-associated adhesin that may function in host colonization by S. pyogenes.

Cloning and characterization of heavy and light chain genes encoding the FimA-specific monoclonal antibodies that inhibit Porphyromonas gingivalis adhesion

FimA of Porphyromonas gingivalis, a major pathogen in periodontitis, is known to be closely related to the virulence of these bacteria and has been suggested as a candidate for development of a vaccine against periodontal disease. In order to develop a passive immunization method for inhibiting the establishment of periodontal disease, B hybridoma clones 123-123-10 and 256-265-9, which produce monoclonal antibodies (Mabs) specific to purified fimbriae, were established. Both mAbs reacted with the conformational epitopes displayed by partially dissociated oligomers of FimA, but not with the 43 kDa FimA monomer. Gene sequence analyses of full-length cDNAs encoding heavy and light chain immunoglobulins enabled classification of the genes of mAb 123-123-10 as members of the mVh II (A) and mVκ I subgroups, and those of mAb 256-265-9 as members of the mVh III (D) and mVκ I subgroups. More importantly, 50 ng/mL of antibodies purified from the culture supernatant of antibody gene-transfected CHO cells inhibited, by approximately 50%, binding of P. gingivalis to saliva-coated hydroxyapatite bead surfaces. It is expected that these mAbs could be used as a basis for passive immunization against P. gingivalis-mediated periodontitis.

A proteomic approach for exploring biofilm in Streptococcus mutans.

Biofilm formation by Streptococcus mutans is considered as its principal virulence factor, causing dental caries. Mutants of S. mutans defective in biofilm formation were generated and analyzed to study the collective role of proteins in its formation. Mutants were characterized on the basis of adherence to saliva-coated surface, and biofilm formation. The confocal laser microscopy and scanning electron microscopy images showed that the control biofilms had cluster of cells covered by layer of exo-polysaccharide while the biofilms of mutants were thin and spaced. Two-dimensional protein electrophoresis data analysis identified 57 proteins that are either up (44 proteins) or down (13 proteins) regulated. These data points to the importance of up and down regulated proteins in the formation of biofilm in Streptococcus mutans.

Pili of oral Streptococcus sanguinis bind to salivary amylase and promote the biofilm formation

Streptococcus sanguinis is a member of oral streptococci and one of the most abundant species found in oral biofilm called dental plaque. Colonization of the oral streptococci on the tooth surface depends on the adhesion of bacteria to salivary components adsorbed to the tooth surface. Recently, we identified unique cell surface long filamentous structures named pili in this species. Herein, we investigated the role of S. sanguinis pili in biofilm formation. We found that pili-deficient mutant, in which the genes encoding the three pilus proteins PilA, PilB and PilC have been deleted, showed an impaired bacterial accumulation on saliva-coated surfaces. Confocal microscopic observations suggested that the mutant was incapable of producing typical three-dimensional layer of biofilm. Ligand blot analysis showed that the ancillary pilus proteins PilB and PilC bound to human whole saliva. Additional analysis demonstrated that PilC bound to multiple salivary components, and one of which was found to be salivary α-amylase. These results indicate that pilus proteins are members of saliva-binding proteins of oral S. sanguinis, and suggest the involvement of pili in its colonization on saliva-coated tooth surfaces and in the human oral cavity.

Experiments on in vivo biofilm formation and in vitro adhesion of Candida species on polysiloxane liners

Microorganisms may colonise polysiloxane soft liners leading to bio-deterioration. The aim of this study was to investigate in vitro adhesion and in vivo biofilm formation of Candida species on polysiloxane surfaces. The materials used in this study were Molloplast B, GC Reline soft, Mollosil Plus, Silagum Comfort and Palapress Vario. The in vitro retention of clinical isolates of Candida albicans to the relining and denture-base materials by microscopic (scanning electron microscopy, SEM), conventional culturing methods and antimicrobial properties of these materials were studied. Candida found on materials and mucosa following long-term use were identified and quantified, and biofilms covering the surfaces were investigated by SEM. There was a significant decrease in the number of cells attached in vitro to saliva-coated surfaces compared with non-treated surfaces. An oral Candida carriage of 78% was found. Candida albicans, C. glabrata, C. intermedia and C. tropicalis were identified. In vivo biofilm formation on the liners appeared as massive colonisation by microorganisms. The results of the in vitro experiments suggest that salivary film influences early colonisation of different C. albicans strains. The film layer also minimises the differences among different strains. The Candida carriage of these patients was similar to denture-wearing patients without soft liners.

Molecular force probe measurement of antigen I/II–matrix protein interactions

Viridans streptococci possess a family of immunologically and structurally related cell-surface proteins, termed antigen I/II, which may function as adhesins and enable oral streptococci to adhere to saliva-coated surfaces and matrix proteins. Here we used atomic force microscopy in the molecular force mode to measure the specific interaction forces between antigen I/II and two matrix proteins, collagen and fibronectin. These matrix proteins provide important binding sites for adherence of oral streptococcal in dentinal caries and endocarditis, respectively. Antigen I/II-coated cantilever tips were brought into contact with collagen- or fibronectin-coated silica coverslips. For the protein I/II-fibronectin interaction experiments, the mean strength of the last ruptures was 216 pN, with most of the detachments located around 125 pN. In antigen I/II-collagen interaction experiments, the mean strength of the last rupture forces corresponded to 136 pN, with the most frequent unbinding force around 75 pN. Thus, our findings definitely suggest that, under the present experimental conditions, antigen I/II binds more strongly to fibronectin than to type I collagen. This might be of relevance for the attachment of viridians streptococci to surfaces exposed to strong hydrodynamic shearing forces under in vivo conditions.

Streptococcus gordonii Modulates Candida albicans Biofilm Formation through Intergeneric Communication

The fungus Candida albicans colonizes human oral cavity surfaces in conjunction with a complex microflora. C. albicans SC5314 formed biofilms on saliva-coated surfaces that in early stages of development consisted of approximately 30% hyphal forms. In mixed biofilms with the oral bacterium Streptococcus gordonii DL1, hyphal development by C. albicans was enhanced so that biofilms consisted of approximately 60% hyphal forms. Cell-cell contact between S. gordonii and C. albicans involved Streptococcus cell wall-anchored proteins SspA and SspB (antigen I/II family polypeptides). Repression of C. albicans hyphal filament and biofilm production by the quorum-sensing molecule farnesol was relieved by S. gordonii. The ability of a luxS mutant of S. gordonii deficient in production of autoinducer 2 to induce C. albicans hyphal formation was reduced, and this mutant suppressed farnesol inhibition of hyphal formation less effectively. Coincubation of the two microbial species led to activation of C. albicans mitogen-activated protein kinase Cek1p, inhibition of Mkc1p activation by H(2)O(2), and enhanced activation of Hog1p by farnesol, which were direct effects of streptococci on morphogenetic signaling. These results suggest that interactions between C. albicans and S. gordonii involve physical (adherence) and chemical (diffusible) signals that influence the development of biofilm communities. Thus, bacteria may play a significant role in modulating Candida carriage and infection processes in the oral cavity.

Identification of novel LPXTG-linked surface proteins from Streptococcus gordonii

Surface adhesion plays an essential part in the survival of the commensal organism Streptococcus gordonii in the oral cavity as well as during opportunistic infections such as endocarditis. At least two types of cell surface protein involved in adhesion are found on the surface of Gram-positive bacteria: those anchored via an LPXTG motif by the enzyme sortase A (SrtA) and those associated with the cell surface by, as yet, unknown mechanisms. In srtA(-) mutants, LPXTG-containing proteins have been shown to be released rather than cross-linked to the cell wall. We have therefore used 2D gel electrophoresis of released proteins from an srtA(-) mutant as well as the wild-type strain, followed by peptide identification by MS, to identify a set of novel proteins predicted to be present on the surface of S. gordonii DL1. This includes two large LPXTG-linked proteins (SGO_0707 and SGO_1487), which both contain tandemly repeated sequences similar to those present in known fibrillar adhesins. A 5'-nucleotidase and a protein with a putative collagen-binding domain, both containing LPXTG motifs, were also identified. Anchorless proteins with known chaperone, stress response and elongation factor functions, apparently responsible for bacterial binding to keratinocytes and saliva-coated surfaces in the absence of the LPXTG-linked adhesins, were also associated with the cell surface. These data reveal a range of proteins to be present on the S. gordonii DL1 cell surface, the expression of which plays an important role in adhesion to epithelia and which represent likely candidates for novel virulence factors in S. gordonii.

In vitro evaluation of yoghurt starter lactobacilli and Lactobacillus rhamnosus GG adhesion to saliva‐coated surfaces

The aim of the study was to evaluate the adhesion of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus rhamnosus strain GG to saliva-coated surfaces in vitro. Fifteen radiolabeled dairy L. delbrueckii subsp. bulgaricus strains and L. rhamnosus GG were tested for their ability to adhere to saliva-coated hydroxyapatite beads and polystyrene microtiter plates and the radioactivity was measured by liquid scintillation counter. The effects of lysozyme on the adhesion of lactobacilli and of pretreatment with lactobacilli on the adhesion of Streptococcus sanguinis were also assessed. All strains tested adhered to saliva-coated surfaces but with significantly different binding frequencies. The adhesion of the L. delbrueckii subsp. bulgaricus strains remained lower in comparison to L. rhamnosus strain GG. One L. delbrueckii subsp. bulgaricus strain showed binding frequency comparable to S. sanguinis. Lysozyme pretreatment of the samples significantly increased lactobacillus adhesion to saliva-coated surfaces. The present results showed significant variations in the adhesion capacity of the Lactobacillus strains studied. Adhesion to oral surfaces is of primary importance for bacterial colonization in the mouth. Only one of the L. delbrueckii subsp. bulgaricus dairy starter culture strains investigated had a high adhesion percentage. This strain might then be considered for further investigations in the oral environment.

Novel effect of plant lectins on the inhibition ofStreptococcus mutansbiofilm formation on saliva-coated surface

Dental caries is caused by the disturbance in oral homeostasis, marked by a notable increase in the population of Streptococcus mutans. Lectins are a group of plant proteins that are capable of recognizing the glycoconjugates present on the bacterial surface. The aim of this study was to evaluate the effect of seven plant lectins on the growth and initial adhesion of S. mutans. Lectins of different carbohydrate specificities were isolated from plant sources by conventional methods of protein purification. The effect on growth of S. mutans was evaluated following CLSI guidelines. None of the lectins used in this study inhibited the bacterial growth and multiplication. The adherence and biofilm formation of bacteria to saliva-coated polystyrene plates was tested in the presence of plant lectins. All the plant lectins tested, inhibited both the adherence and biofilm in a concentration dependent manner. Confocal microscopy and scanning electron microscopy were employed to assess the biofilm formation in the presence of plant lectin (glucose/mannose-specific) at sub-minimal inhibitory concentrations. These evaluations revealed that lectins inhibited the clumping and attachment of S. mutans. Lectins tested here inhibited initial biofilm formation by S. mutans. Glucose/Mannose-specific lectin altered the adhesion arrangement of the bacteria on the saliva-coated surfaces. The plant lectins used in this study may offer a novel strategy to reduce development of dental caries by inhibiting the initial adhesion and subsequent biofilm formation of S. mutans.

Immunogenicity of a Cholera Toxin B Subunit Porphyromonas gingivalis Fimbrial Antigen Fusion Protein Expressed in E. coli

The gram-negative anaerobic oral bacterium Porphyromonas gingivalis initiates periodontal disease through fimbrial attachment to saliva-coated oral surfaces. To study the effects of immunomodulation on enhancement of subunit vaccination, the expression in E. coli and immunogenicity of P. gingivalis fimbrial protein (FimA) linked to the C-terminus of the cholera toxin B subunit (CTB) were investigated. Complementary DNAs encoding the P. gingivalis 381 fimbrillin protein sequence FimA1 (amino acid residues 1-200) and FimA2 (amino acid residues 201-337) were cloned into an E. coli expression vector downstream of a cDNA fragment encoding the immunostimulatory CTB. CTB-FimA1 and CTB-FimA2 fusion proteins synthesized in E. coli BL21 (DE3) cells were purified under denaturing conditions by Ni2+-NTA affinity column chromatography. Renaturation of the CTB-FimA1 and CTB-FimA2 fusion proteins, permitted identification of CTB-FimA pentamers and restored CTB binding activity to GM1-ganglioside to provide a biologically active CTB-FimA fusion protein. Mice orally inoculated with purified CTB-FimA1 or CTB-FimA2 fusion proteins generated measurable FimA1 and FimA2 IgG antibody titers, while no serum fimbrial IgG antibodies were detected when mice were inoculated with FimA1 or FimA2 proteins alone. Immunoblot analysis confirmed that sera from mice immunized with CTB linked to FimA1 or FimA2 contained antibodies specific for P. gingivalis fimbrial proteins. In addition, mice immunized with FimA2 or CTB-FimA2 generated measurable intestinal IgA titers indicating the presence of fimbrial antibody class switching. Further, mice orally immunized with CTB-FimA1 generated higher IgA antibody titers than mice inoculated with FimA1 alone. The experimental data show that the immunostimulatory molecule CTB enhances B cell-mediated immunity against linked P. gingivalis FimA fusion proteins, in comparison to immunization with FimA protein alone. Thus, linkage of CTB to P. gingivalis fimbrial antigens can increase subunit vaccine immunogenicity to provide enhanced protection against periodontal disease.

Autoinducer 2 Is Required for Biofilm Growth ofAggregatibacter(Actinobacillus)actinomycetemcomitans

Autoinducer 2 (AI-2) is required for the growth of Aggregatibacter (Actinobacillus) actinomycetemcomitans in culture under conditions of iron limitation. However, in vivo this organism thrives in a complex multispecies biofilm that forms in the human oral cavity. In this report, we show that adherent growth of A. actinomycetemcomitans on a saliva-coated surface, but not planktonic growth under iron-replete conditions, is defective in a LuxS-deficient background. Biofilm growth of the luxS mutant exhibited lower total biomass and lower biofilm depth than those for the wild-type strain. Normal biofilm growth of the luxS mutant was restored genetically by introduction of a functional copy of luxS and biochemically by addition of partially purified AI-2. Furthermore, introduction of S-adenosylhomocysteine hydrolase, which restores the metabolism of S-adenosylmethionine in the absence of LuxS, into A. actinomycetemcomitans did not complement the luxS mutation unless AI-2 was added in trans. This suggests that AI-2 itself is required for biofilm growth by A. actinomycetemcomitans. A biofilm growth deficiency similar to that of the LuxS-deficient strain was also observed when a gene encoding the AI-2-interacting protein RbsB or LsrB was inactivated. Biofilm formation by A. actinomycetemcomitans was virtually eliminated upon inactivation of both rbsB and lsrB. In addition, biofilm growth by wild-type A. actinomycetemcomitans was reduced in the presence of ribose, which competes with AI-2 for binding to RbsB. These results suggest that RbsB and LsrB function as AI-2 receptors in A. actinomycetemcomitans and that the development of A. actinomycetemcomitans biofilms requires AI-2.