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.     

Immunochemical and functional studies of Actinomyces viscosus T14V type 1 fimbriae with monoclonal and polyclonal antibodies directed against the fimbrial subunit.

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.     

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.     

A polysaccharide from Streptococcus sanguis 34 that inhibits coaggregation of S. sanguis 34 with Actinomyces viscosus T14V.

Coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34 depends on interaction of a lectin on A. viscosus T14V with a cell surface carbohydrate on S. sanguis 34. This carbohydrate was isolated, and its chemical makeup was established. The carbohydrate remained attached to S. sanguis 34 cells through extraction with Triton X-100 and treatment with pronase. It was cleaved from the cell residue by autoclaving and purified by differential centrifugation and column chromatography on DEAE-Sephacel and Sephadex G-75. The polysaccharide contained phosphate which was neither inorganic nor monoester. Treatment with NaOH-NaBH4, followed by Escherichia coli alkaline phosphatase, or with 48% HF at 4 degrees C, followed by NaBH4, yielded inorganic phosphate and oligosaccharide alditols. Therefore, the polysaccharide is composed of oligosaccharide units joined together by phosphodiester bridges. The structure and stereochemistry of the main oligosaccharide alditol was established previously (F. C. McIntire, C. A. Bush, S.-S. Wu, S.-C. Li, Y.-T. Li, M. McNeil, S. Tjoa, and P. V. Fennessey, Carbohydr. Res. 166:133-143). Permethylation analysis, 1H and 31P nuclear magnetic resonance studies on the whole polysaccharide revealed the position of the phosphodiester linkages. The polysaccharide is mainly a polymer of (6) GalNAc(alpha 1-3)Rha(beta 1-4)Glc(beta 1-6)Galf(beta 1-6)GalNAc(beta 1- 3)Gal(alpha 1)-OPO3. It reacted as a single antigen with antiserum to S. sanguis 34 cells and was a potent inhibitor of coaggregation between A. viscosus T14V and S. sanguis 34. Quantitative inhibition of precipitation assays with oligosaccharides, O-allyl N-acetylgalactosaminides, and simple sugars indicated that specific antibodies were directed to the GalNAc end of the hexasaccharide unit. In contrast, coaggregation was inhibited much more effectively by saccharides containing betaGalNAc. Thus, the specificity of the A. viscosus T14V lectin is strikingly different from that of antibodies directed against the S. sanguis 34 polysaccharide.     

Dominant low responsiveness in the IgG response of mice to the complex protein antigen type 1 fimbriae from Actinomyces viscosus T14V.

Type 1 fimbriae from Actinomyces viscosus T14V, composed of a complex protein of Mr 65,000, mediate the adherence of A. viscosus T14V to the host, whereas type 1 fimbriae-specific antibodies inhibit adherence. Genetic control of the serum IgG response to type 1 fimbriae was evaluated in a series of inbred, hybrid, recombinant inbred, and back-cross mice. Mice were given i.p. injections of 10(8) A. viscosus T14V cells in saline on days 0 and 14, and IgG anti-type 1 fimbriae in sera obtained on day 26 were measured by ELISA. Segregation analysis of the responses of (BALB/cJ x A/J)F1 x A/J backcross mice suggested polygenic control. Linkage analysis in (BALB/cJ x A/J)F1 x A/J backcross and SWXL recombinant inbred mice suggested control by genes linked with H-2 and with Ly-17 and Akp-1. In several F1 hybrid strains derived from H-2-disparate high and low responder parental strains, low responsiveness was dominant. The F1 derived from the H-2-identical high and low responder strains CBA/J and C3H/HeJ was a low responder, suggesting that dominant low responsiveness was mediated by non-H-2-linked genes. A three-gene model is proposed for regulation of the type 1 fimbriae response, including an MHC-linked gene, a gene linked with Ly-17 and Akp-1 on the telomeric portion of chromosome 1, and a background gene whose linkage is unknown.     

Effects of oxygen on the growth and metabolism of Actinomyces viscosus

Abstract Actinomyces viscosus is a predominant microorganism in dental plaque. It is, just as the oral Streptococcus spp., a saccharolytic and aero-tolerant organism. We have investigated the effects of oxygen on the growth and metabolism of A. viscosus . To this end A. viscosus Ut 2 was grown in a glucose limited chemostat culture on a chemically defined medium ( D = 0.2 h⁻¹) with exposure to variable amounts of oxygen. The Y_glucose increased from 62.5 g · mol⁻¹ under anaerobic conditions to 149 g · mol⁻¹ under aerobic conditions, while, concomitantly, the carbon recovery from acidic fermentation products decreased from 75% to 7%. Addition of [¹⁴C]glucose to the chemostat showed that the glucose, which was not converted to acidic fermentation products, was instead converted to carbon dioxide or used for the production of biomass. Under aerobic and anaerobic conditions identical cytochrome spectra, containing only two cytochrome b -type absorption bands, were found. It was concluded that electron transport phosphorylation probably occurs both under aerobic and anaerobic conditions. Anaerobically, fumarate served as the electron acceptor, while the high growth yields observed under aerobic conditions are likely to be explained by citric acid cycle activity coupled to electron transport phosphorylation.     

Characterization and cellular localization by monoclonal antibodies of the 60 kDa mannose specific lectin of human promyelocytic cells,HL60

Myelomonocytic lineage cells express anM _r 60 000 mannose specific lectin, MR60 (Pimpaneauet al. (1991),Carbohydr Res 213: 95–108). Under non-reducing conditions, this protein migrates as a 120 000 protein. MR60 does not contain anyN-glycan moiety cleavable by the action ofN-glycanase. MR60 induces a sugar selective aggregation of beads coated with glycosylated albumin: beads bearing -d-mannosyl residues are aggregated while beads bearing -d-glucosyl residues are not. A monoclonal antibody Lec101B, specific for MR60, recognizes a singleM _r 60 000 protein by Western blotting. This monoclonal antibody does not label the cell surface of cells expressing MR60, but decorates intracellular vesicles upon permeabilization of these cells.     

Detection of private and recurrent idiotopes on natural anti-tubulin antibodies by monoclonal anti-idiotopic antibodies

Two monoclonal anti-idiotopic antibodies (anti-Id) were raised in mice against a human monoclonal IgA,K displaying a monospecific anti-tubulin (anti-alpha- and anti-beta-tubulin) activity. One anti-Id (IgG,K) recognized a private idiotope, TID 3.2, present only on the IgA,K immunogen, close to or within the antigen-combining site. The other anti-Id (IgM,K) recognized a recurrent idiotope, TID 7.1, outside the paratope and present in normal human and BALB/c mouse serum, on 2 of 11 polyspecific human monoclonal immunoglobulins and on 6 of 11 murine natural monoclonal auto-antibodies exhibiting a widespread anticytoskeletal protein-binding activity. Both the idiotopes were absent on two induced anti-tubulin antibodies exhibiting a monospecific anti-alpha- and anti-beta-tubulin specificity. Utilizing competitive and additivity immunoassays, we could show that the polyspecific human and mouse anticytoskeletal antibodies tested, whether bearing the TID 7.1 Id or not, appeared to compete in variable degrees for epitopes on the tubulin molecule recognized by the monoclonal IgA,K but distinct from the epitopes recognized by the induced monospecific anti-tubulin antibodies. The high incidence of the recurrent TID 7.1 idiotope in man and mouse suggests an important physiologic and perhaps regulatory function of this idiotope. Furthermore our data suggest that a restricted family of germ-line genes, highly conserved during phylogeny, may encode for these idiotope-bearing Ig molecules.     

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)     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

Summary The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with poreine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.Supported by the Deutsche Forschungsgemeinschaft, Carvas SFB 90     

Mitogenic activity of Actinomyces viscosus. I. Effects on murine B and T lymphocytes, and partial characterization.

Actinomyces viscosus homogenate (AVIS) contins substance(s) which cause spleen cells from conventional and germfree mice to undergo increased DNA synthesis. This mitogenic effect is primarily on B cells since spleen cells from nude mice or T-depleted spleen cells from conventional mice respond as strongly as conventional (T + B) spleen cells. Mouse thymocytes do not respond mitogenically to AVIS. It is unlikely that the mitogenic acitivity is due to the presence of LPS, since A. viscosus is Gram-positive and is not known to have an LPS cell wall component. Also, AVIS is not inactivated by polymyxin B, as are some preparations of LPS, and C3H/HeJ mouse splenocytes respond strongly to AVIS but not to LPS. The activity is heat stable, is not lost upon dialysis, and is not affected by lysozyme. Mitogenic activity is partially lost when AVIS is digested with nonspecific bacterial protease or treated with metaperiodate. Sodium hydroxide treatment completely abolishes mitogenic activity. Actinomycotic lesions are characterized by a long-tern inflammatory response involving a dense plasma cell infiltrate. We suggest that B cell mitogens form Actinomyces may play a role in the elicitation of the plasma cell component of these lesions.     

Detection of glucagon in pancreatic A-cells by monoclonal antibodies

The production of a mouse monoclonal antibody from a hybrid myeloma and its use for the detection of glucagon in tissue sections is reported. The hybrid clone isolated after fusion of mouse myeloma cells with hyperimmune spleen cells from a mouse previously immunized with porcine glucagon allowed us a standardized and permanent source of monoclonal antibodies in a culture cell system. The monoclonal antibody (3 GL 31) specifically reacts with pancreatic A-cells in several species including pig, rabbit, tupaia belangeri and sheep. No immunoreactivity is observed against gut cells and neurons.     

Detection of masked anti-DNA antibodies in lupus sera by a monoclonal anti-idiotype

We report here the use of a monoclonal anti-idiotype 3I to human anti-DNA antibodies to detect in serum idiotype-positive antigen-binding antibodies lacking DNA-binding activity as measured by conventional antigen binding assays. We studied paired serum samples from 13 patients with systemic lupus obtained at two times in the course of their disease: in each patient, one serum sample has anti-DNA activity and the second serum sample has no anti-dsDNA activity detectable by Millipore filter, ELISA, or Crithidia assay. Reactivity with 3I as detected with a radioimmunoassay (RIA) was present in all 13 sera with anti-dsDNA activity. Six patients showed a decrease in 3I reactivity to normal levels in the second serum sample, in which anti-dsDNA antibodies were not detectable by conventional antigen-binding assays. The other seven patients' second serum sample continued to show elevated 3I reactivity by RIA even though no anti-dsDNA activity was apparent. When the 3I-reactive antibodies from these latter patients' sera were eluted from a 3I affinity column, they revealed DNA-binding activity. Furthermore, dsDNA binding by these sera was apparent when they were displayed on Western blots of isoelectric focusing gels run in 8 M urea and incubated with radiolabeled dsDNA. These results indicate that the 3I anti-idiotype can detect anti-DNA antibodies in some sera of SLE patients that lack anti-DNA activity by ordinary assays. These antibodies may be inhibited in binding dsDNA by excess antigen or autologous anti-idiotype, and their DNA binding activity can be unmasked by procedures promoting immune complex dissociation.     

Detection of catalytic monoclonal antibodies.

Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its Km) and reduce its concentration to pseudo-first-order conditions (S(O) much less than Km) so that the signal observed in the presence of a catalytic antibody (delta Pcat) is significantly higher than that of the background (delta P(uncat)). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with p-nitrophenyl ester-hydrolyzing antibodies.     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

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.