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At least 577 records · Page 32Linked to original sources
PubMed1985

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.

Actinomyces↗
PubMed1987

Cloning and expression of a type 1 fimbrial subunit of Actinomyces viscosus T14V.

The type 1 fimbriae of Actinomyces viscosus mediate the adherence of this organism to saliva-treated hydroxyapatite. The gene encoding a putative subunit of this fimbrial adhesin was cloned in Escherichia coli, and its product was examined. A. viscosus T14V chromosomal DNA was partially restricted with Sau3AI and cloned into E. coli JM109 by using the plasmid vector pUC13. Two clones, each containing a different DNA insert with a common 4.1-kilobase region, reacted in colony immunoassays with specific polyclonal as well as monoclonal antibodies directed against A. viscosus T14V type 1 fimbriae. Western blot analysis revealed the expression of a 65-kilodalton protein that migrated slightly behind an antigenically similar protein from native type 1 fimbriae. Deletion analysis showed that the gene encoding the cloned protein was localized on a 1.9-kilobase PstI-BamHI fragment and that transcription was dependent on the lac promoter of the vector. The cloned fimbrial protein was purified from the E. coli cytoplasmic fraction by ion-exchange, immunoaffinity, and gel permeation chromatography. Rabbit antibodies prepared against the cloned protein and against purified A. viscosus type 1 fimbriae gave similar patterns with partially dissociated type 1 fimbriae after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The data therefore provide evidence that the gene cloned encodes a subunit of this fimbrial adhesin.

Actinomyces↗
PubMed1988

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.

Actinomyces↗
PubMed1988

Cloning and nucleotide sequence of a gene for Actinomyces naeslundii WVU45 type 2 fimbriae.

A genomic library of Actinomyces naeslundii WVU45 DNA in Escherichia coli was screened for antigen expression with rabbit antibody against A. naeslundii fimbriae. Western blotting (immunoblotting) of one recombinant clone carrying a 13.8-kilobase-pair insert revealed a 59-kilodalton (kDa) immunoreactive protein. A protein of similar electrophoretic mobility was detected from the isolated fimbrial antigen. Expression of the 59-kDa cloned protein in E. coli was directed by a promoter from the insert. The DNA sequence of the subunit gene was determined, and an open reading frame of 1,605 nucleotides was identified which was preceded by a putative ribosome-binding site and followed by two inverted repeats of 14 and 17 nucleotides, respectively. The reading frame encoded a protein of 534 amino acids (calculated molecular weight, 57,074), and the N-terminal sequence resembled that of a signal peptide. The presence of a 32-amino-acid signal peptide was indicated by amino-terminal sequencing of the fimbriae from A. naeslundii. The sequence, as determined by Edman degradation, was identical to that deduced from the DNA sequence beginning at predicted residue 33 of the latter sequence. Moreover, the amino acid composition of the predicted mature protein was similar to that of the isolated fimbriae from A. naeslundii. Thus, the cloned gene encodes a subunit of A. naeslundii fimbriae.

Actinomyces↗
PubMed1991

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.

Actinomyces↗
PubMed1991

Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.

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PubMed1997

The Arcanobacterium (Actinomyces) pyogenes hemolysin, pyolysin, is a novel member of the thiol-activated cytolysin family.

Arcanobacterium (Actinomyces) pyogenes, an animal pathogen, produces a hemolytic exotoxin, pyolysin (PLO). The gene encoding PLO was cloned, and sequence analysis revealed an open reading frame of 1,605 bp encoding a protein of 57.9 kDa. PLO has 30 to 40% identity with the thiol-activated cytolysins (TACYs) of a number of gram-positive bacteria. The activity of PLO was found to be very similar to those of other TACYs, except that it was not thiol activated. The highly conserved TACY undecapeptide is divergent in PLO; in particular, the cysteine residue required for thiol activation has been replaced with alanine. However, mutagenesis of the alanine residue to cysteine did not confer thiol activation on PLO, suggesting a conformational difference in the undecapeptide region of this toxin. Specific antibodies against purified, recombinant PLO completely neutralized the hemolytic activity of A. pyogenes, suggesting that this organism produces a single hemolysin. Furthermore, these antibodies could passively protect mice against lethal challenge with A. pyogenes, suggesting that like other TACYs PLO is an important virulence factor in the pathogenesis of this organism.

Actinomyces↗
PubMed1998

The Arcanobacterium (Actinomyces) pyogenes plasmid pAP1 is a member of the pIJ101/pJV1 family of rolling circle replication plasmids.

The 2.4-kb plasmid pAP1 from Arcanobacterium (Actinomyces) pyogenes had sequence similarity within the putative replication protein and double-stranded origin with the pIJ101/pJV1 family of plasmids. pJGS84, a derivative of pAP1 containing a kanamycin resistance gene, was able to replicate in Escherichia coli and Corynebacterium pseudotuberculosis, as well as in A. pyogenes. Detection of single-stranded DNA intermediates of pJGS84 replication suggested that this plasmid replicates by the rolling circle mechanism.

Actinomyces↗
PubMed2000

Characterization of the fructosyltransferase gene of Actinomyces naeslundii WVU45.

Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of D-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated from Actinomyces naeslundii WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5' from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving ftf expression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimal ftf expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that ftf was constitutively transcribed. Thus, the A. naeslundii FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.

Actinomyces↗
PubMed1969

Characterization of Actinomyces israelii serotypes 1 and 2.

In a previous serological study, we compared 14 isolates of Actinomyces israelii serotype 2 with 13 serotype 1 cultures. The present study reports the morphological, physiological, and biochemical characteristics of these same 27 cultures. All of the isolates exhibited similar cellular morphology, and all but one produced the typical spider type microcolony on solid media. Twelve of 13 serotype 1 isolates produced the molar tooth type macrocolony, whereas only 2 of 14 serotype 2 cultures produced this type of rough colony. All of the serotype 1 isolates fermented arabinose with the production of acid; none of the serotype 2 cultures fermented this carbohydrate. All 27 cultures produced the greatest amount of growth when cultured under anaerobic conditions and grew poorly or not at all in air. Both groups of organisms produced similar reactions on other biochemical test media; these findings suggested that A. israelii serotype 2 should not be given a species designation.

Actinomyces↗
PubMed1975

Identification of Actinomyces viscosus from canine infections.

Actinomyces viscosus is a gram-positive, non-acid-fact, facultative, catalase-positive, filamentous, or diphtheroidal microorganism. It was isolated from six canine infections during a period of 1.5 years. The organism was cultured from exudate and flaky granules aspirated from infectious granulomas and empyemas. All cultures grew well aerobically and anaerobically with the addition of 10% carbon dioxide. They fermented lactose, produced catalase and acetylmethylcarbinol, reduced nitrates, hydrolyzed aesculin, and did not produce gelatinase or urease. These physiological characteristics distinguish A. viscosus from other morphologically similar organisms.

Actinomyces↗
PubMed1980

Acid-fast actinomyces in a child with pulmonary actinomycosis.

Open-lung biopsy in a child with chronic pneumonia revealed branched gram-positive acid-fast organisms, later identified as Actinomyces israelii. Like Nocardia asteroides, A israelii can be acid fast by the commonly used Putt stain. The pneumonia was cured with 12 weeks of penicillin treatment.

Actinomyces↗
PubMed1988

Identification of Actinomyces (Corynebacterium) pyogenes with the API 20 Strep system.

A total of 62 strains of Actinomyces pyogenes (previously Corynebacterium pyogenes) were examined by the API 20 Strep system (API System, La Balme Les Grottes, Montalieu-Vercieu, France). The system was shown to be reliable and rapid when the tests were compared with standard identification methods. No confusion occurred with streptococcal profiles in the current API 20 Strep data base.

Actinomyces↗
PubMed2000

Infection of a hip prosthesis by Actinomyces naeslundii.

We present the case of a 77-year-old woman who developed an Actinomyces naeslundii infection of a hip prosthesis. The isolate grew well aerobically with 5% CO(2). Possible diagnostic problems may arise in the microbiological laboratory because aerobic growth is not sufficiently accounted for in some of the traditional identification schemes and commercial test kits. Therefore, besides presenting an unusual pathogen in this setting, this report focuses on possible diagnostic problems in the microbiological laboratory.

Actinomyces↗
PubMed1977

Malar mass due to Actinomyces odontolyticus.

Actinomyces odontolyticus was isolated from a patient with a soft tissue mass in the malar region. The organism was identified on the basis of morphological, cultural, and biochemical characteristics. On histological examination, the tissue mass contained several granulomatous foci with small, basophilic staining areas resembling microscopic sulfur granules. This is believed to be the first reported case of actinomycosis due to A. odontolyticus.

Actinomyces↗
PubMed1978

Quantitative fluorescent immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus.

Optimal conditions for a fluorescence immunoassay of antibodies to, and surface antigens of, Actinomyces viscosus ATCC 19246 are described. In the standard fluorescence immunoassay, 10(8) colony-forming units of A. viscosus reacted with an antibody preparation, were washed, and then were treated with an excess of fluorescein-conjugated goat anti-rabbit immunoglobulin G. After another set of washes, fluorescence was determined in a spectofluorometer; in most cases excitation was at 485 nm, with emission measured at 525 nm. These conditions minimized interference from light scatter and stray light. Under appropriate conditions, antibodies to A. viscosus could be readily determined, with the fluorescence of the specific antibody-treated cells more than five times the fluorescence of controls treated with normal rabbit serum. Organisms coated with specific antibody could be detected at levels approaching 10(5) colony-forming units per ml. The standard fluorescence immunoassay procedure was readily adapted to the measurement of either particulate or soluble surface antigens of A. viscosus by competition of the antigen with a fixed amount of antibody in the standard assay system; the competition resulted in an antigen dose-dependent inhibition of fluorescence. The fluorescent immunoassay system thus appears to be a general one that could be applied to other microbial systems as well.

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