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Groups of mice fed diets high in sucrose or glucose were orally inoculated with 10(10), 10(9) or 10(8) colony-forming units of one of the following Actinomyces naeslundii strains possessing the type 1 (T1+) and/or the type 2 (T2+) fimbriae: T14VJ1 (T1+, T2+), 5519 (T1+), 5951 (T2+), and 147 (non-fimbriated). Ninety-six hours after inoculation their upper jaws were cultured to look at the implantation of each of these strains on the teeth. In mice fed a sucrose diet, regardless of the presence or absence of fimbriae, each bacterial strain colonized 100% of the mice at the highest inoculation doses of the infecting organism. But at a dose of 10(8), T14V-J1 was the only strain which colonized 100% (12/12) of the mice, 5519 colonized 10/11, 5951 colonized 9/11 and 147 colonized 7/11. These differences were not statistically significant. When mice were fed a high-glucose diet, 100% infection was achieved with strains T14V-J1, 5519 and 5951 only at the highest dose of 10(10) colony-forming units. Strain 147 colonized in 8/9 of the mice at that dosage. At lower dosages, no bacterial strain implanted in 100% of the mice. In the glucose experiment at a dose of 10(8), strains expressing the T1 fimbriae implanted significantly better than strains without the T1 fimbriae. At a dose of 10(9) colony-forming units, the parent strain T14V-J1 implanted significantly better than strains without the T1 fimbriae. Similarly, strain 5519 (T1+) implanted significantly better than 5951 and implanted better than 147, although the difference was not significant. These results suggest that while the presence of the T1 and T2 fimbriae may confer some advantage in the establishment of these organisms in vivo, even the strains without fimbriae were able to colonize. Strains T14VJ1 and 5519 were found to bind well to hydroxyapatite treated with mouse saliva, while strains 5951 and 147 did not. Only T2 fimbriated strains T14V-J1 and 5951 exhibited a lactose-reversible coaggreation with indigenous strains of enterococci that may contribute to the elevated levels of colonization of strain 5951 in vivo.

A monoclonal antibody to Actinomyces naeslundii (A. viscosus) T14V-J1 type 1 fimbriae, capable of inhibiting the adherence of this bacterium to salivary proline-rich protein-treated hydroxyapatite, was generated by immunization of SWR mice with A. naeslundii 55-19, a strain derived from T14V-J1 that possess only type 1 fimbriae. Supernatants of hybridomas were screened for reactivity with purified type 1 fimbriae. An IgG monoclonal antibody, 86-49E, blocked the adsorption of the parent strain to proline-rich protein-treated hydroxyapatite by 77% with 1.0 microgram/ml of the monoclonal antibody; the Fab fragment derived from this monoclonal antibody inhibited adherence by 38% at the same concentration. Similarly, the adherence of strain 55-19 was inhibited by 100% and 64% to proline-rich protein-treated hydroxyapatite with 1.0 micrograms/ml of IgG and Fab fragments respectively. Control monoclonal antibody to the subunit of type 1 fimbriae, as well as to Actinobacillus actinomycetemcomitans caused only minimal adherence inhibition. Monoclonal antibody 86-49E also agglutinated both type 1 fimbriae-bearing strains of A. naeslundii T14V-J1 and 55-19 but not strains 59-51 and 147, which lack type 1 fimbriae. Further confirmation of the specificity of monoclonal antibody 86-49E was obtained using these fimbria-deficient mutant strains in an enzyme-linked immunosorbent assay, with the monoclonal antibody binding only to strains possessing type 1 fimbriae. Immunogold labeling in conjunction with electron microscopy suggested binding of monoclonal antibody 86-49E occurring near the distal end of the fimbriae. In contrast, when a monoclonal antibody specific for the type 1 fimbrial subunit but not capable of adherence inhibition was used together with 86-49E in double-labeling experiments, extensive labeling of the fimbriae by the subunit antibody was noted. These data suggest that a monoclonal antibody specific for the type 1 fimbriae of A. naeslundii that is capable of binding to a discrete site on the fimbriae has the capacity to inhibit the adsorption of this organism to saliva-treated hydroxyapatite.

Our earlier studies demonstrated that clinical strains of Prevotella intermedia, isolated from human periodontal pockets, possess various types of fimbria (surface appendages) as determined ultrastructurally. These bacteria have the ability to agglutinate selected mammalian erythrocytes. Hemagglutinating activity exhibited by these cells may be attributable to these surface structures. Strain 17, which possess fimbriae of 8 nm in diameter and readily agglutinates human, monkey, sheep, rabbit, and mouse erythrocytes was selected to determine whether these fimbriae possessed the hemagglutinating activity. Fimbriae were mechanically sheared, concentrated by ammonium sulfate precipitation, solubilized in 10 mM Tris-HCl buffer containing 0.5% deoxycholate and partly purified by ultracentrifugation in a 10-50% linear sucrose gradient. Isolated fimbriae banded at a density of 1.20-1.15 g/ml, appeared fairly uniform ultrastructurally, and possessed hemagglutinating activity. The hemagglutinating activity of P. intermedia whole cells and isolated fimbriae was reduced by treatment with proteases and eliminated by treatment with heat at 80 degrees C for 10 min. The optimal pH for the hemagglutination was 7.0. In the process of hemagglutination, P. intermedia whole cells and isolated fimbriae bound to rabbit erythrocytes as observed by: (a) decrease in the hemagglutinating activity of bacterial whole cells and isolated fimbriae after incubation with rabbit erythrocytes, (b) increase in hemagglutinating activity of the red cells used for absorption, and (c) the presence of P. intermedia whole cells and fimbriae on red cells after absorption as revealed by immunoelectron microscopy. Both the immune immunoglobulin G of the rabbit polyclonal anti-fimbriae antibody and the immune immunoglobulin G and its Fab fragments of the murine monoclonal anti-fimbriae antibody were effective inhibitors of hemagglutination mediated by isolated fimbriae. Immunogold labeling showed that the monoclonal antibody bound specifically to P. intermedia fimbriae. These results collectively suggest that the hemagglutination exhibited by P. intermedia may be attributable to the fimbriae or the fimbrial-associated components.

Each of five monoclonal antibodies (mAbs) prepared against the type 1 fimbriae of Actinomyces viscosus T14V reacted with a 54 kDa cloned protein previously identified as a fimbrial subunit. This purified protein completely inhibited the reaction of a specific anti-type-1-fimbria rabbit antibody with A. viscosus whole cells. Maximum values for the number of antibody molecules bound per bacterial cell ranged from 7 x 10(3) to 1.2 x 10(4) for the different 125I-labelled mAbs and was approximately 7 x 10(4) for 125I-labelled rabbit IgG or Fab against either type 1 fimbriae or the 54 kDa cloned protein. Although the different mAbs, either individually or as a mixture, failed to inhibit the type-1-fimbria-mediated adherence of A. viscosus T14V to saliva-treated hydroxyapatite, each rabbit antibody gave 50% inhibition of adherence when approximately 5 x 10(4) molecules of IgG were bound per cell. However, binding of each corresponding rabbit Fab had no significant effect on bacterial attachment unless much higher concentrations were used. These findings suggest that antibodies directed solely against the 54 kDa fimbrial subunit do not react with the putative receptor binding sites of A. viscosus T14V type 1 fimbriae. Instead, inhibition of attachment by the polyclonal antibodies may depend on an indirect effect of antibody binding that prevents the fimbria-receptor interaction.

Antibodies reactive with type 1 and type 2 fimbriae from Actinomyces viscosus T14V specifically inhibit the adherence of A. viscosus T14V to salivary pellicle-coated tooth surfaces and other bacteria, and these antibodies are thought to modulate colonization by this microorganism. These studies were done to determine whether previously noted differences in the antibody responses of inbred mice to type 1 and type 2 fimbriae might be under genetic control. The serum immunoglobulin G (IgG) and IgM antibody responses of inbred, F1 hybrid, and H-2 congenic mice, immunized with A. viscosus T14V cells, were analyzed by enzyme-linked immunosorbent assays for antibodies reactive with A. viscosus T14V whole-cell type 1 and type 2 fimbriae. The results confirmed earlier findings and indicated striking variations in the amounts of IgG anti-type 1 (23-fold) and anti-type 2 (48-fold) fimbria antibodies elicited. The responses of the 17 inbred strains tested showed a relatively continuous distribution from high to low, as well as marked differences in the responses of H-2 and Igh-C identical strain pairs. An analysis of the responses of F1 hybrid and H-2 congenic mice indicated dominance of the low-responder gene(s) and control by H-2-linked genes. Antisera from two high-responder strains inhibited in vitro bacterial adherence to a much greater degree than antisera from a low-responding strain. These data suggest polygenic control of the magnitude of the IgG anti-type 1 and anti-type 2 fimbria antibody responses by H-2-linked genes as well as background genes not associated with H-2 or Igh-C loci.

Salivary proline-rich proteins (PRPs), which were purified from parotid saliva, were adsorbed onto 15-nm-diameter gold particles to visualize specific binding of the salivary molecules to Actinomyces viscosus type 1 fimbriae. Negatively stained preparations incubated with PRP-gold conjugates but not bovine serum albumin-gold complexes bound specifically to bacteria possessing type 1 fimbriae, A. viscosus T14V-J1 and 5519. Binding of the PRP-gold probes to strains deficient in type 1 fimbriae, i.e., strains 5951 (type 2 fimbriae only) and 147 (no fimbriae), was negligible.

Fimbria-specific antibody responses were compared in mice immunized with purified fimbrial adhesins in the region of the submandibular gland (i.e., local site) or at a remote site in the back. One hundred micrograms of fimbriae isolated from Actinomyces viscosus T14V was used as the vaccine. Four subcutaneous injections of the vaccine in the local site induced greater amounts of fimbria-specific immunoglobulin G (IgG) in serum and saliva than three injections. However, there was no difference in the response of fimbria-specific IgA in serum and saliva. Fimbria-specific IgG in serum and saliva were first detected 21 days after the primary immunization at both the local or remote sites. Fimbria-specific IgA in serum was first detected 28 days after the primary immunization at both the local or remote sites. However, fimbria-specific IgA in saliva occurred only in mice immunized with the fimbrial vaccine at the local site and was first detected 14 days after the primary immunization. Both serum and saliva from mice immunized 4 times with the fimbrial vaccine in the local site inhibited in vitro adsorption of strain T14V cells to hydroxyapatite beads pretreated with normal mouse saliva, whereas adsorption of strain T14V cells suspended in serum and saliva from sham-immunized animals was not inhibited. Collectively, these data suggest that mice immunized locally in the submandibular gland region with a vaccine composed of purified fimbrial adhesins provide a potential model for evaluating the efficacy of fimbria-specific antibodies in saliva to inhibit strain T14V colonization of tooth surfaces.

Mutants of Actinomyces viscosus T14V lacking type 1 or type 2 fimbriae or both were selected by their failure to react with rabbit antibodies against either or both fimbrial antigens. Immunospecific double labeling with iron dextran and ferritin-conjugated antibodies showed two types of fimbriae on individual cells of the parent organism, a single type on mutant strains with type 1+2- and type 1-2+ fimbriae and no labeled or unlabeled fimbriae on a type 1-2- fimbria-deficient strain. The mutational loss of one fimbrial antigen did not appear to affect expression of the other, since bacteria with one or two types of fimbriae bound similar amounts of a monoclonal antibody directed against the fimbrial antigen present on both bacterial phenotypes. The strong adsorption of strains with type 1+2+ or 1+2- fimbriae to saliva-treated hydroxyapatite and weak adsorption of those with type 1-2+ or no fimbriae was consistent with the known involvement of type 1 fimbriae in this attachment process. Similarly, the A. viscosus lectin was clearly associated with the expression of type 2 fimbriae, since only the strains with type 1+2+ or 1-2+ fimbriae participated in lactose-sensitive coaggregations with Streptococcus sanguis 34. Further studies using the fimbria-deficient mutant strains showed that aggregation of A. viscosus T14V in the presence of sialidase-treated human saliva involved both types of fimbriae, whereas neither type was required for the lactose-resistant coaggregation of the organism with certain streptococcal strains.

Specific antibodies and their Fab fragments were used to study adsorption of Antinomyces viscosus T14V to saliva-treated hydroxyapatite. Antibody against A. viscosus T14V blocked adsorption of the strain to saliva-treated hydroxyapatite, and this activity was removed after preincubation of the immune immunoglobulin G (IgG) with purified type 1 fimbriae in amounts near equivalence but not after preincubation with type 2 fimbriae in antigen excess. Specific IgG or Fab fragments against type 1 fimbriae inhibited adsorption of strain T14V to saliva-treated hydroxyapatite and promoted desorption of cells already attached to the apatite surface. Anti-type 2 IgG or Fab fragments did not possess these activities. These data demonstrate that type 1 but not type 2 fimbriae mediate adsorption of strain T14V to the saliva-treated hydroxyapatite surface.

Actinomyces viscosus T14V-J1 and its fimbria-deficient mutant strain possessing type 1 fimbriae strongly aggregated with latex beads treated with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative. These type 1+ strains did not aggregate with latex beads treated with other proteins, such as salivary amylase, salivary histidine-rich polypeptides, laminin, type 1 collagen, fibronectin, or C1q. The type 1+ strains also adsorbed well to experimental pellicles formed with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative on hydroxyapatite (HA) surfaces. These interactions were inhibited with immunoglobulins and Fabs specific for type 1 fimbriae. Type 1- actinomyces exhibited feeble adsorption to latex beads or HA treated with any of the aforementioned proteins. Collectively, these data indicate that actinomyces type 1 fimbriae may specifically interact with several proline-rich salivary molecules, forming experimental pellicles on HA or polystyrene surfaces.

Use of specific fimbria-defective mutants derived from Actinomyces viscosus T14 has shown that salivary acidic proline-rich protein 1 and statherin serve as receptors for type 1 fimbriae which mediate attachment of the organism to experimental pellicles on apatitic surfaces.

We studied the adsorption, morphological, and serological characteristics of selected Actinomyces and related species. Evaluation of uranyl acetate-stained cells by electron microscopy revealed wide variations among strains in the frequency of surface fimbriae. These variations did not always correlate with the percent adsorption to saliva-treated hydroxyapatite of the various Actinomyces strains. However, two strains of Rothia dentocariosa possessing no surface fimbriae and five strains of A. israelii possessing very few surface fimbriae exhibited feeble adsorption to saliva-treated hydroxyapatite. Although the calculated number of adsorption sites on saliva-treated hydroxypatite did not vary widely among the strains tested, significant differences were observed in the affinities calculated for some species or serotypes. The mean affinities for strains of A. viscosus serotype 2 and A. naeslundii serotype 3 were similar, and these strains adsorbed well to saliva-treated hydroxyapatite. The mean adsorption and affinity for the A. naeslundii strain serotype 1 and all strains of A. israelii tested were significantly less than those determined for the A. viscosus serotype 2 or A. naeslundii serotype 3 strains. Adsorption inhibition activity of antiserum to strain T14V, previously shown to be solely related to antibodies in immune serum directed against the VA1 fimbria (fibril) antigen, was removed by preadsorption of the antiserum with most A. viscosus and A. naelundii strains, but not with A. israelii strains. This suggests some cross-reactivity among strains of A. viscosus and A. naeslundii but not A. israelii. Adsorption to saliva-treated hydroxyapatite of all A. viscosus and A. naeslundii strains tested was strongly inhibited by fimbriae isolated from A. viscosus strain T14V. Collectively, these data suggest that the adsorption of certain A. viscosus and A. naeslundii strains is mediated by surface fimbriae, many of which appear serologically cross-reactive with strain T14V fimbriae.

Experiments were performed to determine whether immunization of mice with fimbrial adhesins isolated from Actinomyces viscosus T14V could modulate infection of tooth surfaces in animals challenged with the homologous strain. Saliva and sera from animals immunized in the submandibular gland region contained elevated levels of fimbria-specific immunoglobulin A (IgA) and IgG, whereas saliva and sera from sham-immunized animals did not. There was a statistically significant inverse correlation between the presence of fimbria-specific antibodies in saliva and serum and the levels of bacterial colonization on molar tooth surfaces. These results suggest that fimbrial adhesins may effectively modulate infection of tooth surfaces by periodontopathic bacteria.

Monospecific antibody against the type-1 fimbriae of Actinomyces viscosus T14V reacted with 12 strains of A. viscosus (serotype 2), 12 strains of A. naeslundii (serotype 2 or serotype 3), but not with 11 serotype 1 strains of A. naeslundii. All strains positive for the type-1 fimbrial antigen adsorbed strongly to saliva-treated hydroxyapatite, and in many instances (18 of 24 strains) this interaction was inhibited by the monospecific antibody. These findings strongly suggest that only those strains of actinomyces which possess type-1 fimbriae adhere to the tooth surfaces, and that these fimbriae constitute the principal adhesin involved in this interaction.