Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.     

The cell wall-associated levansucrase of Actinomyces viscosus.

Actinomyces viscosus produces both a soluble extracellular levansucrase and a cell wall-associated levansucrase. The enzyme from cell walls was solubilized by lysozyme digestion. The soluble extracellular and cell wall-associated forms of the enzyme were compared and appeared to be identical, based on molecular weight estimations, kinetic parameters, and reactions with antisera. The product of both forms of the enzyme was a high molecular weight, branched levan, as shown by its reactivity with myeloma proteins specific for beta(2 leads to 1) and for beta(2 leads to 6) linkages in fructosans. Although levansucrase remained tightly bound to the levan which it synthesized, the enzyme did not bind to exogeneously added levan. Regarding the potential pathogenicity of the levan product, pure levan, produced using purified levansucrase, did weakly activate complement by the alternative pathway. However, the pure levan did not directly cause bone resorption in an in vitro bone resorption assay.     

Identification of Human Strains of Actinomyces viscosus

Catalase-positive actinomycetes which closely resemble the “hamster organism” described by Howell have been isolated from dental calculus and other human sources. These cultures could not be distinguished from the hamster strains on the basis of morphology, oxygen requirements, biochemical reactions, or cell wall composition. These human isolates have been classified with the hamster strains as Actinomyces viscosus. The strains from hamster and human sources fell into two serotypes. Serotype 1 contains the hamster strains plus one strain of unknown origin, whereas serotype 2 contains all of the human strains.     

Characterization of the binding of Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) to glycosphingolipids, using a solid-phase overlay approach

Actinomyces naeslundii (ATCC 12104) and Actinomyces viscosus (ATCC 19246) were radiolabeled externally (125I) or metabolically (35S) and analyzed for their ability to bind glycosphingolipids separated on thin layer chromatograms or coated in microtiter wells. Two binding properties were found and characterized in detail. (i) Both bacteria showed binding to lactosylceramide (LacCer) in a fashion similar to bacteria characterized earlier. The activity of free LacCer was dependent on the ceramide structure; species with 2-hydroxy fatty acid and/or a trihydroxy base were positive, while species with nonhydroxy fatty acid and a dihydroxy base were negative binders. Several glycolipids with internal lactose were active but only gangliotriaosylceramide and gangliotetraosylceramide were as active as free LacCer. The binding to these three species was half-maximal at about 200 ng of glycolipid and was not blocked by preincubation of bacteria with free lactose or lactose-bovine serum albumin. (ii) A. naeslundii, unlike A. viscosus, showed a superimposed binding concluded to be to terminal or internal GalNAc beta and equivalent to a lactose-inhibitable specificity previously analyzed by other workers. Terminal Gal beta was not recognized in several glycolipids, although free Gal and lactose were active as soluble inhibitors. The binding was half-maximal at about 10 ng of glycolipid. A glycolipid mixture prepared from a scraping of human buccal epithelium contained an active glycolipid with sites for both binding specificities.     

Hydrophobic interactions of group A streptococci with hexadecane droplets.

The adherence of Streptococcus pyogenes cells to hexadecane droplets was measured by vortexing water suspensions of streptococci with hexadecane. It was found that adherence of the organisms to hexadecane droplets was abolished by pretreating the organisms with trypsin, pepsin at pH 4.5, or HCl solutions at 95 degrees C. Streptococcal adherence was best expressed in organisms harvested during the stationary phase of growth and was inhibited by fatty acid-free albumin because of the interaction of the protein with the streptococcal surfaces. The data suggest that adherence to hexadecane droplets measures the availability on the surface of S. pyogenes cells of lipophilic residues that are either hydrophobic regions of surface protein structures or, more likely, glycolipids complexed with and oriented by surface proteins.     

Actinomyces viscosus cell-free synthesis of extracellular slime polysaccharide

A cell-free extract of Actinomyces viscosus T14Av catalyzed the synthesis of extracellular N-acetylglucosamine-rich slime polysaccharide. The activity was localized in the cytoplasmic membrane fraction and required the presence of ADP-glucose and UDP-N-acetylglucosamine. Maximal activity was demonstrated at pH 7.5 and also required the presence of divalent cations such as Mg2+ or Mn2+. Extracellular slime appeared to serve as a primer for slime biosynthesis. The antibiotic tunicamycin acted as an inhibitor of slime formation. However, another glucosamine analogue, amphomycin, as well as the antibiotic bacitracin produced moderate stimulatory effects on slime biosynthesis.     

Surface localization of sialic acid on Actinomyces viscosus

This study reports the presence of sialic acid in Actinomyces viscosus strains T14V and T14AV. Mild acid hydrolysis of whole organisms released a compound which reacted positively in the periodate-thiobarbituric acid, direct Ehrlich's and resorcinol assays, and which co-chromatographed on paper with authentic N-acetylneuraminic acid. Strain T14V contained 10-fold greater concentrations of sialic acid than did strain T14AV. Sialic acid content was dependent upon the stage of growth of the culture, reaching a maximum in early stationary phase. Epifluorescence microscopy of fluorescein isothiocyanate (FITC)-conjugated Limulus polyphemus agglutinin (LPA), a lectin specific for sialic acid, revealed a uniform distribution of bound lectin on the surfaces of strains T14V and T14AV. Additional evidence for surface localization was obtained by demonstration of whole-cell agglutination of both strains with LPA. All LPA interactions with A. viscosus were inhibited by the presence of 0.1 M-N-acetylneuraminic acid. Neuraminidases from Clostridium perfringens, Arthrobacter ureafaciens and Vibrio cholerae did not release detectable amounts of sialic acid, but the extracellular enzyme from A. viscosus cleaved amounts equivalent to those obtained by acid hydrolysis. Other laboratory strains (W1053, M100, W859, 5-5S, RC45, ATCC 19246, and 'binder') as well as recent clinical isolates of A. viscosus were agglutinated by LPA and released sialic acid upon mild acid hydrolysis. Surface-available sialic acid has been implicated in the inhibition of alternative complement pathway activation and subsequent opsonophagocytosis. Thus the occurrence of surface sialic acid in A. viscosus may represent a mechanism of pathogenesis for this oral bacterium.     

Kinetics of adherence of Actinomyces viscosus to saliva-coated silica and hydroxyapatite beads

Adherence of 14C-labelled strains of Actinomyces viscosus to uncoated and saliva-coated silica and hydroxyapatite beads had both loose and firm components, probably reflecting different subpopulations of bacteria within a single culture. Adherence was characterized by the proportion of bacteria available for each type of adherence and a constant (Kb) for each combination of bacterial strain and bead surface. Loose adherence, which was greater with silica than with hydroxyapatite beads, always involved many more bacteria than firm adherence. Firm adherence was greater with A. viscosus WVU627 than A. viscosus TF11. The association rate constants (Ka) for loose and firm adherence were similar, indicating simultaneous processes, but the dissociation rate constant (Kd) was lower for loose adherence than for firm adherence. Removal of loosely adhering bacteria by washing may only reflect their distance from the bead surface. Silica beads were convenient for studying bacterial adherence and formed an acceptable coating of salivary glycoprotein.     

Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.

Microorganisms in dental plaque live in constant association with saliva. The role of saliva in the adherence of bacteria to the teeth and the antibacterial properties of saliva have been well investigated; less interest has been shown in the possible role of saliva as a substrate for oral microorganisms. In this study it was shown that saliva can serve as a growth medium for oral Streptococcus spp. and Actinomyces viscosus. The cell production of these organisms on saliva was carbohydrate limited. The doubling times for growth on glucose-supplemented saliva (4 to 5 mmol/liter) ranged from 1.6 to 4.0 h. The availability of carbohydrate sources for the oral microflora is discussed in relation to microbial growth in the oral cavity.     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei.

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Actinomyces viscosus strain GA: characteristics of its cellular biological retardant(s)

Actinomyces viscosus strain GA produces an exocellular biological retardant(s) that prevents certain vegetable plants from becoming overgrown. The biological retardant(s) was assayed using the etiolated wheat coleoptile assay, and fractionation of culture supernatant fluid resulted in a partial purification of the retardant(s). The biological retardant(s) was most active around pH 7 in the bioassay and when applied to sterile soil mixture. The biological retardant(s) was tentatively identified as a derivative of a rare hexose carbohydrate (but not an amino sugar) but an exact structure was not determined. In a sterilized synthetic soil system, the biological retardant(s) has an effect on tomato cultivars similar to that observed by the synthetic plant growth regulators Alar (succinic acid 2,2-dimethylhydrazide) and Bonzi (paclobutrazol).     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Isolation of a neuraminidase gene from Actinomyces viscosus T14V.

A genomic library of Actinomyces viscosus T14V DNA in lambda gt11 was screened for expression of neuraminidase activities. Four recombinant clones were detected that gave blue fluorescence upon incubation with a fluorogenic substrate, 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Of these, two were identical, and all of the neuraminidase-positive clones shared a common 3.4-kbp DNA region. Expression of the enzyme activities in Escherichia coli carrying the cloned DNA was independent of the lacZ promoter of the vector. Maxicell analysis revealed that the 3.4-kbp DNA insert directed synthesis of a protein with an apparent molecular mass of 100,000 Da. The protein from cell extracts of E. coli clones migrated as a single band that stained for enzyme activity after electrophoresis in a nondissociating polyacrylamide gel. Moreover, human erythrocytes incubated previously with cell lysates from neuraminidase-positive E. coli were hemagglutinated by Actinomyces spp. The enzyme expressed by E. coli was active on substrates containing alpha-2,3 and alpha-2,6 ketosidic linked sialyl residues. Similar substrate specificities were obtained for both the extracellular and cell-associated neuraminidases from A. viscosus T14V. The 3.4-kbp insert hybridized to DNA fragments in a Southern blot containing A. viscosus T14V chromosomal DNA that had been digested with various restriction endonucleases. Data from hybridization studies show that A. viscosus T14V contains a single copy of the neuraminidase gene.     

Properties of sialidase isolated from Actinomyces viscosus DSM 43798

The cell-bound sialidase of Actinomyces viscosus DSM 43798 was solubilized by mechanical cell disruption and lysozyme treatment. The enzyme was enriched 30,000-fold by cation-exchange chromatography, gel-filtration, and FPLC ion-exchange chromatography, thus obtaining 10 micrograms sialidase protein from 26 g wet cells with a specific activity of 680 U/mg protein. Since sialidase activity was also found in the culture medium, this enzyme was isolated as well, requiring the additional application of FPLC gel-filtration. Both sialidase preparations were apparently homogenous on SDS-PAGE and have similar properties. The substrate specificity of the A. viscosus sialidase was tested with 16 sialoglycoconjugates: The enzyme showed a higher activity with serum glycoproteins than with gangliosides, mucins or sialyllactoses. 4-O-Acetylated N-acetylneuraminic acid was not cleaved from equine submandibular gland mucins or serum glycoproteins in contrast to N-acetyl- and N-glycoloylneuraminic acid. 9-O-Acetyl-N-acetylneuraminic acid was released from bovine submandibular gland mucin, as confirmed by TLC. The sialidase hydrolyses alpha(2----6)-linkages more rapidly than alpha(2----8)- and alpha(2----3)-bonds. Cations, except Hg2+, or chelating agents have no influence on enzyme activity. The sialidase has a relatively high molecular mass of 150 kDa, but consists of only one unit. The enzyme is labile towards freezing and thawing, but can be stored at 4 degrees C in 0.1 M acetate buffer, pH 5.     

Application of polymerase chain reaction with arbitrary primer (AP-PCR) to strain identification of Porphyromonas (Bacteroides) gingivalis

Several molecular methods are currently available for identification and discrimination of bacterial strains within the same species, which vary in efficiency and required labour. Here we applied a novel method for fingerprinting genomes, called arbitrarily primed PCR (AP-PCR), to the delineation of strains within the species Porphyromonas gingivalis. Using a single primer on a set of nine strains, nine simple distinct banding patterns, indicative of genetic polymorphism, were observed. Common amplicons and amplicons shared by only some strains were also observed, the latter suggesting that AP-PCR can be used to generate polymorphic markers. Genomic fingerprinting obtained by AP-PCR was independent of the quality of DNA. Assays performed directly using whole cells as a source of DNA template indicated that AP-PCR from colony is a quick, simple and accurate procedure.     

Expression of Actinomyces viscosus antigens in Escherichia coli: cloning of a structural gene (fimA) for type 2 fimbriae.

A cosmid gene library of Actinomyces viscosus T14V was prepared in Escherichia coli to examine the expression of A. viscosus antigens and to gain insight into the structure of A. viscosus type 1 and type 2 fimbriae. Out of this library of 550 clones, 28 reacted in a colony immunoassay with antibodies against A. viscosus cells. The proteins responsible for these reactions were identified in three clones. Clones AV1209 and AV2009 displayed nonfimbrial antigens with subunits of 40 and 58 kilodaltons, respectively. Clone AV1402 showed a 59-kilodalton protein that reacted with monospecific antibody against type 2 fimbriae and that comigrated with a subunit of type 2 fimbriae during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that AV1402 expresses a gene (fimA) for a subunit of A. viscosus type 2 fimbriae.     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of surface fibrils from taxonomically typical Actinomyces viscosus WVU627.

Fibrils of Actinomyces viscosus WVU627 (numerical taxonomy cluster 1) were obtained by homogenization and purified by ultrafiltration, ammonium sulfate precipitations, gel filtration, and ion-exchange chromatography. Electron microscopy and resolution of a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis attested to the purity of the preparation. Purified fibrils were composed mainly of protein; small quantities of carbohydrate and phosphorus were detected. Immunoelectrophoresis revealed only a single precipitable antigen, which migrated slightly toward the anode, in reactions between purified fibrils and antiserum raised against either whole bacterial cells or the purified fibrils themselves. Immunoelectron microscopy with ferritin-conjugated antifibril antibody hemagglutination inhibition, and bacterial agglutination tests demonstrated that fibrils of Actinomyces viscosus cluster 1 strains shared some common antigens with clusters 2, 3, 4 and 6, but did not cross-react with typical Actinomyces naeslundii of cluster 5. Stability tests revealed that after heat or alkali treatment, the fibrils lost their antigenicity and disappeared from electron micrographs. They were affected less by sodium dodecyl sulfate, sonic, or acid treatments.     

Sodium dodecyl sulfate potentiates collagen degradation by proteases from Porphyromonas (Bacteroides) gingivalis

Abstract Collagen degradation by protease(s) from Bacteroides gingivalis was estimated by spectrophotometry with insoluble, type I, collagen a substrate. When 1% sodium dodecyl sulfate (SDS) was included in the assay, there was a 7-fold increase in reaction velocity. The protease(s) were extracted from the bacteria with one percent Triton X-100 and partially purified by gel chromatography on Superose HR 12. The SDS-potentiated enzyme (tentatively named proteinase D(odecyl)) eluted immediately after the void volume and migrated at a position corresponding to 100 kDa by localization of enzyme activity after SDS-polyacrylamide gel electroporesis. The temporal degradation of collagen fibrils by proteinase D was illustrated by phase contrast microphotography of fibrils dispersed in 1% SDS and addition of concentrated enzyme from the edge of the coverslip. During 30 min, the diameter of the fibrils gradually diminished, and some fibrils exhibited a zig-zag profile. After 45 min, most of the fibrils had disappeared. Incubation of proteinase D with 1% SDS for 6 h at 37°C did not diminish the activity of the enzyme. The collagen was completely degraded to small peptides when SDS was present.