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PubMed1978

Role of colonization in the virulence of Actinomyces viscosus strains T14-Vi and T14-Av.

Germfree rats fed a high-sucrose diet were inoculated with Actinomyces viscosus strain T14-Vi (virulent) or T14-Av (avirulent). The mean recovery of strain T14-Vi from six extracted finely ground molars of rats sacrificed after 90 days was 1.1 x 10(8) colony-forming units (CFU). The mean recovery of strain T14-Av was 5.7 x 10(7) CFU, which was significantly less. Strain T14-Vi caused severe alveolar bone loss, but only minimal bone loss occurred in rats infected with strain T14-Av. Scanning electron microscopy of teeth of germfree rats revealed that strain T14-Vi colonized in the fissures as well as on tooth surface areas near the gingiva; strain T14-Av also colonized in fissures but was unable to colonize the teeth near the gingiva. In studies with conventional rats fed a high-sucrose diet, streptomycin-resistant strain T14-Vi colonized on the teeth of all rats inoculated with in the order of 10(8) or 10(7) CFU and on the teeth of about half of the rats inoculated with 10(6) or 10(5) CFU. In contrast, streptomycin-resistant strain T14-Av could not be detected on the teeth of any of the rats in groups similarly inoculated. In vitro "resting" cells of both strains suspended in conventional or germfree rat saliva survived to comparable degrees. [(3)H]thymidine-labeled T14-Vi cells adhered well to hydroxyapatite (HA) beads and to HA beads pretreated with saliva obtained from germfree or conventional rats. In contrast, T14-Av cells adhered less well than did T14-Vi cells to HA, whereas their adherence to saliva-coated HA was negligible. Transmission electron microscopy of negatively stained T14-Vi and T14-Av cells repeatedly passed in 1% phosphotungstic acid revealed fibrils on cells of both strains. T14-Av cells were covered by large amounts of extracellular material which was presumably heteropolysaccharide; little extracellular material was present on the surface of T14-Vi cells. T14-Vi cells had a relatively low affinity for the heteropolysaccharide synthesized by strain T14-Av. Other evidence also suggested that this polysaccharide had a relatively low affinity for saliva-coated HA. Collectively, the evidence indicates that the difference in periodontopathic potential between strains T14-Vi and T14-Av results from their different abilities to colonize teeth. This difference is probably due to the lower adherence of T14-Av cells to teeth rather than to their ability to grow in the mouth. The low affinity of T14-Av cells for tooth surfaces may be due, in part, to the presence of large amounts of cell-surface-associated polysaccharide.

Actinomyces↗
PubMed1978

Modification of surface composition of Actinomyces viscosus T14V and T14AV.

The morphology and serology of Actinomyces viscosus T14V and T14AV were compared. When grown in supplemented tryptic soy broth, the virulent strain (T14V) possessed an extensive network of cell surface fibrils. In this medium, the avirulent strain (T14AV) possessed a microcapsule, absent on strain T14V, and a comparatively small number of surface fibrils. Mild acid extraction (Lancefield procedure) solubilized common antigenic components on both strains as well as components detectable only in the virulent strain T14V (virulence-associated antigens 1 and 2). When grown in Socransky chemically defined medium or Carlsson complex medium, the avirulent strain possessed increased amounts of surface fibrils and virulence-associated antigens. Whole cells and extracts of avirulent cells grown in Socransky medium absorbed antibodies to virulence-associated antigens with approximately the same efficiency as did whole cells and extracts of strain T14V, suggesting antigenic similarity between the two cell types. The results strongly support the hypothesis that observable differences between A. viscosus strains T14V and T14AV represent quantitative, rather than qualitative, differences in particular cell surface components. In addition, the magnitude of these differences can be modified by changing growth conditions.

Actinomyces↗
PubMed1979

Model studies on dental plaque formation: deoxyhexoses in Actinomyces viscosus.

A careful examination of two strains of Actinomyces viscosus, T14V (virulent) and T14AV (avirulent), revealed no qualitative or quantitative difference in 6-deoxyhexose content of their cell surface. For a further study of the role of these sugars in cell surface-related phenomena, the stereochemical configuration of deoxyhexoses of A. viscosus T14 was established by two complementary approaches. (i) Examination of the biosynthetic pathway was found to lead to the formation of both 6-deoxy-l-talose and 6-deoxy-l-mannose and showed no differences in the ability of either bacterial strain, A. viscosus T14V or T14AV, to produce the precursors of these cell wall components. The biosynthetic pathway for 6-deoxy-l-talose and 6-deoxy-l-mannose was found to originate from deoxy-thymidine diphosphate (dTDP)-d-glucose, which in turn is converted to dTDP-4-keto-6-deoxy-d-glucose. Epimerization at carbons 3 and 5 of the hexose moiety of dTDP-4-keto-6-deoxy-d-glucose is followed by stereospecific reduction with reduced nicotinamide adenine dinucleotide phosphate to yield dTDP-6-deoxy-l-talose and dTDP-6-deoxy-l-mannose. In cell-free extracts of both A. viscosus T14 and T14AV, an identical ratio of 6-deoxy-l-talose to 6-deoxy-l-mannose of 1:8 was produced. Known precursors for the d-isomers of the same 6-deoxyhexoses such as guanosine diphosphate-d-mannose and dTDP-d-mannose were not converted by A. viscosus T14 cell-free extracts. (ii) Isolation of 6-[U-(14)C]deoxytalose and 6-[U-(14)C]deoxymannose from both strains of A. viscosus T14 was carried out by growing cells in a medium containing d-[U-(14)C]glucose. Again no qualitative or quantitative difference was noticeable between the two strains when 6-deoxy-hexoses were released from whole cells or purified cell walls by acid hydrolysis. Radioactive 6-[U-(14)C]deoxytalose isolated from the cell surface was used in an isotope dilution experiment to establish the stereochemical configuration of this 6-deoxyhexose. The radioactive sugar was mixed with unlabeled standard d- or l-6-deoxyhexose, respectively, and conversion to the corresponding 1-phenylflavazole derivative was carried out. Recrystallization to constant specific activity identified the radioactive sugar isolated from A. viscosus to be the l-isomer. A facile synthesis of the rare sugars 6-deoxy-l-talose and 6-deoxy-d-talose is reported.

Actinomyces↗
PubMed1979

Surface fibrils (fimbriae) of Actinomyces viscosus T14V.

Surface antigens of Actinomyces viscosus T14V were released from cell walls by digestion with lysozyme. These were separated by ion-exchange and gel filtration chromatography into fractions rich in carbohydrate or protein. The former contained a polysaccharide high in 6-deoxytalose, along with a peptide fragment from the cell wall. In the protein-rich fractions, material of high molecular weight was present, which contained some carbohydrate and up to 14.3% nitrogen. Aspartic acid, threonine, glutamic acid, lysine, alanine, and glycine were detected in these fractions, along with smaller amounts of 10 other amino acids. Most of the alanine was present as the L isomer and thus was not from peptidoglycan. Electron microscopy of the high-molecular-weight material revealed long fibrils, 3.5 to 4.5 nm in diameter, which resembled those seen on bacterial cells. V-specific antiserum, prepared by absorbing anti-A. viscosus T14V serum with cell walls of the avirulent strain (A. viscosus T14AV), did not react with the 6-deoxytalose polysaccharide but reacted well with isolated fibrils, and this was not inhibited by 6-deoxytalose.

Actinomyces↗
PubMed1979

Infectious agents in immunodeficient murine models: pathogenicity of Actinomyces israelii serotype I in congenitally athymic (nude) mice.

Nude (Nu/Nu) and heterozygous (NU/+) mice were infected with Actinomyces israelii serotype I by either intranasal instillation or intravenous injection. Lung clearance data and 50% lethal dose values indicated that T-lymphocytes were not necessary for pulmonary clearance or prevention of systemic actinomycosis. However, T-lymphocyte deficiency may play a role in the localized cervicofacial form of actinomycosis.

Actinomyces↗
PubMed1979

Adherence of Actinomyces viscosus T14V and T14AV to hydroxyapatite surfaces in vitro and human teeth in vivo.

Adsorption of Actinomyces viscosus strains T14V and T14AV to hydroxyapatite (HA) surfaces was studied, using an adsorption model based on the Langmuir adsorption isotherm. Data generally followed the adsorption model as judged by high correlation coefficients obtained for both strains to most of the treated surfaces studied. The number of binding sites for strains T14V and T14AV cells to human saliva-treated HA was similar to that for untreated HA. The affinity of strain T14V for saliva-treated HA was tenfold greater than the affinity of strain T14AV for that surface. To approximate the pellicle of the gingival crevice and margin and to determine whether adherence by strain T14V was to specific saliva or serum receptors, experimental pellicles were formed on HA by saliva/serum mixtures. The number of binding sites on the saliva/serum-treated HA remained the same as for the saliva-treated surface. Although the affinity of strain T14V cells for the saliva/serum HA surface remained generally the same as the affinity for the HA treated with saliva alone, the affinity of strain T14AV cells decreased further as the serum content increased. Strain T14V cell numbers adsorbed to serum-treated HA, and albumin-treated HA were less than those adsorbed to saliva-treated HA, indicating that the adherence by strain T14V was to specific saliva receptors. In vivo results from streptomycin-resistant mutants of both strains T14V and T14AV confirmed in vitro results using saliva-serum pellicles. Pretreatment of strain T14V with proteolytic enzymes and heat inhibited adherence to saliva-treated HA, suggesting that the adherence receptor(s) on the cell surface of strain T14V is protein in nature.

Actinomyces↗
PubMed1979

Relationship between host age and susceptibility to oral colonization by Actinomyces viscosus in Sprague-Dawley rats.

The colonization of Actinomyces viscosus strain Ny-1R on the molar teeth of conventional and ex-germfree rats of various ages fed either a high-sucrose diet, a high-glucose diet, or laboratory chow was studied. Conventional rats directly after weaning and up to 30 days of age are less susceptible to experimental infection by strain Ny-1R than are older rats regardless of the test diet. The relationship between host age and susceptibility to infection is also demonstrable in ex-germfree rats fed a high-sucose diet. Host factors responsible for the differences in susceptibility were investigated. The results from these studies do not implicate host antibodies, host indigenous flora, or host saliva. In other studies, it was demonstrated that within the mouths of rats, strain Ny-1R preferentially colonizes in the pits and fissures of the molar teeth rather than on the dorsum of the tongue or on the vestibular mucosa. In short-term experiments, it was found that strain Ny-1R attaches to the first molars of 40-day-old conventional rats to a greater extent than it attaches to the first molars of 20-day-old rats. The differences in attachment and subsequent colonization of strain Ny-1R in 20- and 40-day-old rats may be related to the varying amounts of the reduced enamel epithelium and connective tissue present in the fissures of the molar teeth.

Actinomyces↗
PubMed1980

Immunological properties of Actinomyces viscosus: comparison of blastogenic and adjuvant activities.

A water-soluble extract of Actinomyces viscosus was tested for its capacity to induce deoxyribonucleic acid synthesis in spleen cells from normal mice and to augment the in vitro antibody-forming cell (AFC) response to the T-cell-dependent antigen, sheep erythrocytes (SRBC) by T-cell-deficient adherent spleen cell monolayers from the same mice. Our results indicated that the water-soluble extract induced an in vitro blastogenic response, as assessed by tritiated thymidine incorporation into mouse spleen cells. The water-soluble extract also augmented the antibody response to SRBC. However, after fractionation of the water-soluble extract on Biogel P-300, not all of the fractions induced a blastogenic response and augmented an AFC response to SRBC. This difference in the ability of A. viscosus fractions to induce in vitro responses suggests that the water-soluble extract has multiple immunological properties. Chemical analysis of the water-soluble extract indicated an enrichment for amino acids and amino sugar common to peptidoglycan.

Actinomyces↗
PubMed1980

Fibril-mediated adherence of Actinomyces viscosus to saliva-treated hydroxyapatite.

Fibril-mediated adherence of Actinomyces viscosus strain T14V cells to saliva-treated hydroxyapatite was studied. Fibrils were purified by ammonium sulfate precipitation and differential centrifugation from the crude supernatant of whole cells that were sheared by one passage through a French pressure cell. Purified fibrils and crude supernatant inhibited strain T14V adherence to saliva-treated hydroxyapatite to similar extents. However, anti-strain T14V serum and antifibril specific antibody completely abolished strain T14V adherence. The blocking immunoglobulin could be adsorbed from anti-T14V serum by strain T14V whole cells, by purified fibrils, and, to a lesser extent, by cell walls. It was concluded that fibrils mediate adherence of strain T14V cells to saliva-treated hydroxyapatite. In addition, fibril preparations were shown to contain more than 95% protein and to be antigenically homogeneous by immunodiffusion and Laurell rocket immunoelectrophoresis. Purified fibril preparations showed serological identity with the virulence-associated 1 antigen of Lancefield-extracted T14V cells, whereas crude supernatants contained both virulence-associated 1 and virulence-associated 2 antigens, as shown by rocket immunoelectrophoresis.

Actinomyces↗
PubMed1980

In vitro expression of immunoglobulin M and G subclasses by murine B lymphocytes in response to a polyclonal activator from Actinomyces.

A cell wall extract from the gram-positive bacterium Actinomyces viscosus contains the mitogen AVIS, a potent polyclonal B-cell activator for murine B lymphocytes. Cultures of splenocytes from heterozygous nude mice in the presence of an optimal concentration of AVIS responded by a deoxyribonucleic acid synthesis response, and proliferaction reached maximal levels after 3 to 4 days. There was no requirement for T cells in the deoxyribonucleic acid synthesis, proliferactive, immunoglobulin M (IgM), or IgG responses. Significant numbers of IgM-producing cells were present as early as day 2 of culture, whereas later in the culture periods (days 3 to 6) IgG-producing plasmablasts and plasma cell were observed. In cultures of splenocytes from nude mice stimulated with AVIS for 4 to 5 days, 20 to 25% of the recoverable cells synthesized IgM, and 10% contained only IgG2 or IgG3; 5 to 8% of the cells stained for both IgM and IgG2 or both IgM and IgG3. Fine-structure analysis of AVIS-stimulated splenocytes from heterozygous nude mice after 3 days of culture demonstrated that 20 to 25% of the cells were activated to various degrees. Of most importance, all of the activated cells had the characteristic of B lymphoblasts, plasmablasts, or plasma cells. This is the first demonstration of a polyclonal B-cell activator other than lipopolysaccharide which induces IgG3 synthesis. We suggest that AVIS may be a useful probe for the exploration of the functional activities of subpopulations of B cells.

Actinomyces↗
PubMed1980

Role of bacterial interactions in the colonization of oral surfaces of Actinomyces viscosus.

The effects of several microbial residents of human oral cavities on the attachment of Actinomyces viscosus T14V to tooth surfaces were assessed by using the saliva-coated hydroxyapatite (sHA) in vitro system. Attachment to sHA by A. viscosus T14V was not inhibited in the presence of Streptococcus mutans GS5, either in the presence or absence of sucrose. Precoating sHA beads with S. mutans in the presence of sucrose also did not retard strain T14V attachment. However, the presence of Streptococcus sanguis M5 during strain T14V attachment markedly inhibited the interaction of A. viscosus T14V with sHA, whereas another S. sanguis strain, strain G9B, produced relatively weak inhibition. The inhibitory effect of S. sanguis M5 appeared to result from the direct interaction of this organism with A. viscosus T14V. Conversely, the presence of strain T14V markedly increased the attachment of S. sanguis M5 to sHA while partially inhibiting the attachment of S. sanguis G9B. Moreover, sHA beads precoated with A. viscosus T14V bound much higher levels of S. sanguis M5 relative to uncoated sHA, whereas S. sanguis G9B attachment was only weakly inhibited. It was also observed that sHA beads precoated with either S. sanguis M5 or G9B inhibited subsequent attachment by strain T14V. These results suggest the possibility that the attachment sites for both S. sanguis strains partially overlap those for A. viscosus T14V. Streptococcus mitis weakly inhibited attachment of strain T14V to sHA, and both Streptococcus salivarius and Lactobacillus casei had little effect on attachment. In addition, both a radioisotope attachment assay and fluorescent microscopy demonstrated no significant attachment of A. viscosus T14V to human epithelial cells.

Actinomyces↗
PubMed1980

Attachment of Actinomyces naeslundii to human buccal epithelial cells.

A standardized assay was used to measure the attachment of Actinomyces naeslundii ATCC 12104 to washed human buccal epithelial cells. Treatment of the A. naeslundii cells with hyaluronidases, wheat germ lipase, protease, trypsin, heat, or sonic oscillation significantly reduced their ability to attach to epithelial cells. Treatment of the epithelial cells with the above enzymes did not influence the attachment of A. naeslundii. Extraction of A. naeslundii with NaOH also significantly reduced the ability of the bacterium to attach to human buccal epithelial cells. The neutralized and dialyzed NaOH extract contained both carbohydrate and protein substances in a ratio of about 1:1. Adding this extract back to the extracted bacterial cells partially restored their ability to attach to epithelial cells. When the NaOH extract was preincubated with epithelial cells and residual extract was removed by washing, attachment of normal A. naeslundii was partially blocked. The ability of the extracted material to block attachment was significantly reduced when treated with hyaluronidases or with wheat germ lipase. Treatment with heat, protease, or trypsin did not significantly reduce the ability of the extracted materials to block attachment. Pretreatment of the epithelial cells with hyaluronic acid or chondroitin sulfate also reduced subsequent attachment of normal A. naeslundii cells. Pretreatment of epithelial cells with dextrans, proteins, or unpure mannose did not influence subsequent attachment of A. naeslundii. Pretreatment of A. naeslundii with galactose and lactose significantly inhibited attachment to normal epithelial cells. The results suggest that the attachment of A. naeslundii to human buccal epithelial cells may involve mucopolysaccharides similar to hyaluronic acid on the surface of the bacterial cells. Other attachment mechanisms may also be operative.

Actinomyces↗
PubMed1981

In vitro stimulation of immunoglobulin production from human peripheral blood lymphocytes by a soluble preparation of Actinomyces viscosus.

In this paper, we report the ability of a soluble, ultrasonicate preparation of Actinomyces viscosus to stimulate in vitro immunoglobulin production by human peripheral blood lymphocytes. Immunoglobulin A (IgA), IgG, and IgM concentrations were determined with a highly sensitive and specific micro-enzyme-linked immunosorbent assay (micro-ELISA). A viscosus-induced immunoglobulin production was T lymphocyte dependent and was inhibited by monocytes. The immunoglobulin response was optimal when A. viscosus (1,000 microgram/ml) was removed from the cultures on day 2 of incubation. Antibodies specific for A. viscosus were not detected, indicating that A. viscosus was activating a polycolonal B-lymphocyte response. Since periodontal lesions in humans are characterized by the accumulation of a large number of B lymphocytes, the polyclonal activation of these cells by A. viscosus may affect the severity and resolution of the disease.

Actinomyces↗
PubMed1981

Common antigens of streptococcal and non-streptococcal oral bacteria: immunochemical studies of extracellular and cell-wall-associated antigens from Streptococcus sanguis, Streptococcus mutans, Lactobacillus salivarius, and Actinomyces viscosus.

Soluble extracellular antigens (ESA) were prepared from the culture supernatant of exponential growing cells of Streptococcus sanguis OMZ 9 by a combination of ammonium sulfate precipitation and chromatography on a Bio-Gel P6 column. Soluble cell wall antigens (WEA) were obtained from the bacterial pellet by extraction with 1 M phosphate buffer (pH 6). Antisera against whole cells of S. sanguis and S. mutans of different serotypes, 10% trichloroacetic extracts of bacterial cell walls, dextran, ESA, and WEA were prepared by injecting the different antigens several times in rabbits. ESA and WEA were prepared from a representative strain of Bratthall's seven serological groups, Lactobacillus salivarius, and Actinomyces viscosus. All sera showed various agglutinin titers against heat-killed cells, and titers were generally higher with homologous cells. The comparison of the different antigens using agar gel diffusion and immunoelectrophoresis showed the presence of extracellular common antigens in both ESA and WEA between the different strains. Absorption of anti-ESA sera with WEA, and anti-WEA sera with ESA, showed the existence of a specific antigen common to all bacteria in each fraction. Enzymatic treatment of the antigen before immunodiffusion demonstrated the protein nature of the two antigens present in ESA and WEA.

Actinomyces↗
PubMed1981

Alveolar bone loss in rats after immunization with Actinomyces viscosus.

We investigated a possible cause-and-effect relationship between sensitization against Actinomyces viscosus Nyl and destructive periodontal disease in RIC-Sprague-Dawley rats. Germfree rats (66) were either immunized with A. viscosus Nyl (day 20) or orally infected with A. viscosus Nyl (days 38 and 39) or both. We measured alveolar bone loss in maxillary and mandibular molars, in vitro T-lymphocyte responsiveness, and serum antibody titers. In immunized and monoassociated rats bone loss in both jaws progressed rapidly between days 37 and 72, whereas the rate of further resorption decreased until day 100. In monoassociated rats, development of bone loss was much slower, and the maximum resorption measured was, at best, half of the bone loss compared with the former group. However, no amplification of bone loss by immunization was observed in a second experiment using 63 conventional rats kept in relative gnotobiosis. Antibody titers to A. viscosus Nyl in gnotobiotic monoassociated rats were higher in immunized animals, whereas no difference was found in the respective groups of the relative gnotobiotic experiment. The fact that immunization more than doubled alveolar bone loss in gnotobiotic monoassociated rats confirms the allergic nature of the disease. The lack of such an effect under conventional conditions points to suppressor mechanisms whose decrease might convert stable periodontal lesions into progressive ones.

Actinomyces↗
PubMed1981

Regulation of extracellular slime production by Actinomyces viscosus.

Extracellular slime polysaccharides produced two Actinomyces viscosus strains, T14V and T14AV, were compared. In various media containing glucose, T14Av produced abundant extracellular viscous slime polysaccharide, whereas T14V produced lower levels. Furthermore, fractionation of these polysaccharides showed that the two extracellular polysaccharides differed in molecular size and net charge. Since there was a significant difference in the relative abilities of chemically defined medium and chemically defined tissue culture medium to support slime production by T14Av, the nutritional factors influencing the production of extracellular slime were examined. Sodium bicarbonate was demonstrated to stimulate both cellular growth and the production of extracellular slime. In chemically defined medium with and without sodium bicarbonate, strain T14Av produced large quantities of viscous slime in glucose and sucrose media. In contrast, relatively low levels of slime were produced in fructose, lactose, raffinose, and inositol media, even though sodium bicarbonate stimulated the growth of T14Av in these latter media.

Actinomyces↗
PubMed1981

Effects of metabolic inhibitors on extracellular fructosyltransferase production in Actinomyces viscosus.

Extracellular fructosyltransferase (levansucrase; EC 2.4.1.10) production in Actinomyces viscosus T14AV was demonstrated to occur concomitantly with cellular growth. The inhibition of both cellular ribonucleic acid and protein synthesis resulted in no further accumulation of enzyme activity. The antibiotic sodium clofibrate differentially inhibited the production of fructosyltransferase by strain T14AV. Furthermore, the antibiotic preferentially inhibited [14C]acetate incorporation into cellular lipid, but did not affect protein synthesis. In addition, no inhibition of fructosyltransferase production was observed upon the addition of the fatty acid acid synthesis inhibitor cerulenin. On the other hand, extracellular fructosyltransferase production was apparently stimulated in the presence of the cell wall synthesis inhibitors penicillin, amphomycin, and tunicamycin. These results are discussed in terms of the mechanism of extracellular protein production in A. viscosus.

Actinomyces↗
PubMed1982

Growth rates of Actinomyces viscosus and Streptococcus mutans during early colonization of tooth surfaces in gnotobiotic rats.

Germfree Osborne-Mendel rats were monoassociated with Actinomyces viscosus or Streptococcus mutans. The adherence and subsequent growth of these organisms on the tooth surface was studied by means of total viable cell counts. Both A. viscosus and S. mutans showed a lag phase and an exponential growth phase, similar to logarithmic growth in batch cultures. The exponential growth rates of S. mutans and A. viscosus were 0.63 h-1 (doubling time [td] = 1.1 h) and 0.24 h-1 (td = 2.9 h), respectively. After a period of rapid growth, the rate declined and the populations approached a steady state. The presence of a sucrose-containing diet did not significantly influence the exponential growth rates of A. viscosus and S. mutans, but had a slight negative effect on the initial adherence of S. mutans at the tooth surface.

Actinomyces↗