Cloning of Prevotella intermedia loci demonstrating multiple hemolytic domains.

A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.

Identification of hemolytic activity in Prevotella intermedia.

Hemolysin production was measured in strains of Prevotella intermedia. Zones of beta-hemolysis were detected on agar plates supplemented with either sheep, rabbit or human erythrocytes. A standard tube assay was performed on cell suspensions of the organism to measure hemolytic activity, which was found to be dose dependent, eliminated by heat treatment, and saturable with increasing concentrations of blood. Growth-phase experiments suggested that hemolysin production was increased during logarithmic growth and was reduced during stationary phase. Cell fractionation, performed on several strains of P. intermedia, localized the activity in the outer membrane and in cell vesicles. The biological implication of this study is that P. intermedia, by virtue of its hemolytic activity, is capable of liberating the hemoglobin from erythrocytes, thereby acquiring an essential nutrient, iron, for its metabolism.

Fimbrial-mediated colonization of murine teeth by Actinomyces naeslundii.

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.

Inhibition of adherence of Actinomyces naeslundii (Actinomyces viscosus) T14V-J1 to saliva-treated hydroxyapatite by a monoclonal antibody to type 1 fimbriae.

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.

Isolation and characterization of Actinomyces viscosus mutants defective in binding salivary proline-rich proteins.

Recent studies have provided evidence for human salivary proline-rich proteins (PRPs) serving as potential receptors in the acquired pellicle for Actinomyces viscosus type 1 fimbriae. We report here the isolation of mutants derived from A. viscosus T14V-J1 which are defective in binding to PRPs partially purified from parotid gland saliva. Mutagenesis with ethyl methanesulfonate preceded enrichment for cells nonreactive with PRPs by successive adsorptions with PRP-treated latex beads. Screening was accomplished by random selection of 250 isolated colonies from each of four enrichment cycles and reaction with PRP-treated latex beads in microtiter plates. Two mutants of independent origin were examined for adherence to hydroxyapatite treated with either PRPs, proline-rich glycoproteins, deglycosylated proline-rich glycoproteins, or whole saliva. Additional surface properties that were examined included agglutination with polyclonal antisera to type 1 and type 2 fimbriae, agglutination by a monoclonal antibody to type 1 fimbriae that inhibits adherence of the parent strain to saliva-treated hydroxyapatite, the ability to bind monoclonal antibody to the type 1 fimbrial subunit, and lactose-reversible coaggregation with Streptococcus sanguis 34. Both mutants exhibited reduced binding to hydroxyapatite treated with whole saliva or salivary protein preparations but were still capable of reaction with antiserum to type 1 and type 2 fimbriae. In addition, these mutants possessed the ability to bind monoclonal antibody to the type 1 fimbrial subunit in amounts comparable to the amount bound by the parent strain but were not agglutinated by the adherence-inhibiting monoclonal antibody. When considered with previously published data, these results suggest that an adhesive molecule is probably associated with type 1 fimbriae and allows for the interaction of A. viscosus with constituents in the salivary pellicle.

Subgingival microbiota in squirrel monkeys with naturally occurring periodontal diseases.

The squirrel monkey (Saimiri sciureus) has been proposed as an in vivo model for the study of subgingival colonization by suspected periodontopathogens, such as black-pigmented porphyromonads and prevotellas (BP/P). However, the indigenous microbiota of the squirrel monkey has not been well described. Therefore, in order to more fully characterize the oral microbiota of these animals, we studied two groups of squirrel monkeys from widely different sources. Group I consisted of 50 breeding colony monkeys ranging in age from 9 months to over 6 years which had been raised in captivity; group II consisted of 16 young sexually mature monkeys recently captured in the wild in Guyana. Group I animals in captivity had developed moderate to severe gingivitis, with a mean gingival index (GI) of 2.6; 52% of the sites bled, 26% had detectable calculus, and 83% had detectable BP/P. A group I subset (six animals), for which predominant cultivable microbiota was described, had a mean GI of 2.4. Colony morphology enumeration revealed that five of the six subset animals were detectably colonized with BP/P (range, 0 to 16.9%) and Actinobacillus actinomycetemcomitans (range, 0 to 3.9%); all subset animals were colonized with Fusobacterium species (range, 0.8 to 3.6%), Actinomyces species (range, 2.3 to 11%), and gram-positive cocci (range, 1.4 to 21.4%). Predominant cultivable microbiota results revealed the presence of many bacterial species commonly found in the human gingival sulcus. At baseline, group II animals were clinically healthy and had a mean GI of 1.4; 67% of the sites bled and 2.1% had calculus, and none of the animals had detectable BP/P. Neisseriae were very common in noninflamed sites. Subsequently, when inflamed sites were compared with noninflamed sites in group II animals after they had been maintained in captivity for 6 months, inflamed sites exhibited a more complex microbiota and increased proportions of gram-negative rods and asaccharolytic bacteria.

Genetic control of serum antibody responses of inbred mice to type 1 and type 2 fimbriae from Actinomyces viscosus T14V.

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.

Immune modulation of Prevotella intermedia colonization in squirrel monkeys.

Colonization of the gingival crevice by black-pigmented Porphyromonas or Prevotella spp. (BP/P), including Porphyromonas gingivalis (formerly Bacteroides gingivalis) and Prevotella intermedia (formerly Bacteroides intermedius), is thought to be an important ecological event which may result in the destruction of connective tissues supporting the teeth. Theoretically, periodontal diseases could be prevented if these or other periodontal pathogenic microorganisms did not colonize the subgingival area. The humoral immune response is one mechanism which may modulate bacterial colonization in the gingival crevice. In the present study, we tested the effect of systemic humoral immunity on subgingival colonization by indigenous P. intermedia in squirrel monkeys (Saimiri sciureus). Animals rendered essentially free of detectable BP/P by a single scaling, 10 days of tetracycline therapy, and toothbrushing three times per week were immunized with P. intermedia 1447 or were sham immunized with phosphate-buffered saline. Subsequently, all oral hygiene procedures were discontinued and five teeth in one quadrant were ligated with bacterium-soaked suture material to facilitate BP/P colonization. Immunization resulted in a significant increase in the level of immunoglobulin G anti-P. intermedia antibody in serum. Two weeks after ligation was initiated, P. intermedia could be detected in five of six sham-immunized and three of six immunized animals. Immunization was associated with a reduction in the emergence of indigenous P. intermedia in the gingival crevice.

Pellicle receptors for Actinomyces viscosus type 1 fimbriae in vitro.

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.

Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey.

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.

Antigenic variation of indigenous streptococci.

Isolates of Group D streptococci indigenous to the murine oral cavity were studied to detect the occurrence of antigenic variation. Group D streptococci cultured from molar homogenates of Balb/c mice were randomly selected for study on the basis of distinctive colony morphology. Isolates obtained over a 12-week period were biotyped using the API 20S system, and subjected to Lancefield extraction and rocket immunoelectrophoresis for serotyping. All isolates were compared with an arbitrarily selected standard test strain (W1S-1) isolated the first week of the first experimental series. Four biotypes were encountered during the first week of two experimental series. Two very unusual biotypes detected during the first experimental series persisted throughout that series, as did two more common biotypes throughout the second experimental series. Anti-W1S-1 serum produced three precipitin bands (antigens O, D, and K) against W1S-1 Lancefield extract and against the respective biotypes detected during the first week of the two series. Of the three antigens detected, only the group antigen (D) did not vary during either experimental series. Antigenic variants lacking the O or K antigen and bearing these distinctive phenotypes were repeatedly isolated in subsequent weeks. Ultimately, 16% of 190 strains isolated during the first series and 26% of 167 strains isolated during the second series proved to be antigenic variants of the predominant biotypes detected in both series.

Relative hydrophobicities of Actinomyces viscosus and Actinomyces naeslundii strains and their adsorption to saliva-treated hydroxyapatite.

The present study examined 42 strains of Actinomyces spp. to determine whether adsorption to saliva-treated hydroxyapatite (SHA) of the selected strains of this prominent group of dental-plaque bacteria correlated with hydrophobicity. The relative hydrophobicity of the strains was determined by their adsorption to hydrophobic gels (i.e., phenyl-Sepharose) and their aggregation in ammonium sulfate. Within serogroups the relative hydrophobicity for the strains was similar. The relative adsorption of strains to SHA was also similar within the respective serogroups. Strains which were relatively hydrophobic, as judged by their binding to the hydrophobic gel and aggregation in low concentrations of ammonium sulfate, adsorbed well to SHA. Strains which adsorbed poorly to SHA were relatively hydrophilic since they did not bind well to the hydrophobic gel and were only aggregated in relatively high concentrations of ammonium sulfate. Tween 80, a nonionic detergent known to inhibit hydrophobic interactions, blocked binding of cells to the hydrophobic gel, suggesting that hydrophobic interactions had been inhibited. However, Tween 80 exhibited no influence on the adsorption of cells to SHA. Thus, although there was a strong statistical correlation between the relative hydrophobicity of a strain and its adsorption to SHA, the data were consistent with the view that other interactions, such as ionic bonds and interactions between complimentary macromolecules, are involved in adsorption of the Actinomyces strains to SHA.