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1.  Requirements for invasion of epithelial cells by Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1993;61(4):1239-1245.
Actinobacillus actinomycetemcomitans, an oral bacterium implicated in human periodontal disease, was recently demonstrated to invade cultured epithelial cells (D. H. Meyer, P. K. Sreenivasan, and P. M. Fives-Taylor, Infect. Immun. 59:2719-2726, 1991). This report characterizes the requirements for invasion of KB cells by A. actinomycetemcomitans. The roles of bacterial and host factors were investigated by using selective agents that influence specific bacterial or host cell functions. Inhibition of bacterial protein synthesis decreased invasion, suggesting the absence of a preformed pool of proteins involved in A. actinomycetemcomitans invasion. Inhibition of bacterial and eukaryotic energy synthesis also decreased invasion, confirming that A. actinomycetemcomitans invasion is an active process. Bacterial adherence to KB cells was indicated by scanning electron microscopy of infected KB cells. Further, the addition of A. actinomycetemcomitans-specific serum to the bacterial inoculum reduced invasion substantially, suggesting a role for bacterial attachment in invasion. Many of the adherent bacteria invaded the epithelial cells under optimal conditions. Inhibitors of receptor-mediated endocytosis inhibited invasion by A. actinomycetemcomitans. Like that of many facultatively intracellular bacteria, A. actinomycetemcomitans invasion was not affected by eukaryotic endosomal acidification. These are the first published observations describing the requirements for epithelial cell invasion by a periodontopathogen. They demonstrate that A. actinomycetemcomitans utilizes a mechanism similar to those used by many but not all invasive bacteria to gain entry into eukaryotic cells.
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PMCID: PMC281353  PMID: 8454326
2.  Invasion of epithelial cells by Actinobacillus actinomycetemcomitans: a dynamic, multistep process. 
Infection and Immunity  1996;64(8):2988-2997.
The invasion process of Actinobacillus actinomycetemcomitans, a periodontopathogen, was studied with microscopy and viable quantitative assays using both KB and Madin-Darby canine kidney (MDCK) epithelial cells. Microscopy revealed that the events associated with the A. actinomycetemcomitans invasion process occurred rapidly. Scanning electron micrographs revealed A. actinomycetemcomitans associated with craters on the KB cell surface and others entering the KB cells through apertures with lip-like rims within 30 min of infection. Both transmission electron and immunofluorescence micrographs demonstrated that by this time some bacteria had, in fact, already entered, replicated, and exited host cells. Scanning electron micrographs revealed that infected KB cells exhibited fibrillar protrusions which contained bulges with the conformation of bacteria. Some protrusions formed intercellular connections between KB cells. Immunofluorescence micrographs revealed protrusions which harbored A. actinomycetemcomitans. The spread of internalized A. actinomycetemcomitans from one MDCK epithelial cell monolayer to another was demonstrated using a sandwich assay developed in our laboratory. Transcytosis of A. actinomycetemcomitans through polarized MDCK cells was also demonstrated. This study indicates that soon after entry of A. actinomycetemcomitans bacteria into epithelial cells, they undergo rapid multiplication and may subsequently be found in protrusions which sometimes extend between neighboring epithelial cells. The protrusions are thought to mediate the cell-to-cell spread of A. actinomycetemcomitans. Cell-to-cell spread may also occur by the endocytosis of A. actinomycetemcomitans bacteria which have been released into the medium via rudimentary protrusions which do not interconnect epithelial cells. The finding that the A. actinomycetemcomitans invasion process is so dynamic sheds significant new light on the interaction of this periodontopathogen with mammalian cells.
PMCID: PMC174179  PMID: 8757825
3.  Characteristics of adherence of Actinobacillus actinomycetemcomitans to epithelial cells. 
Infection and Immunity  1994;62(3):928-935.
Actinobacillus actinomycetemcomitans smooth variants [SUNY 75(S), SUNY 465, 652] were investigated for their ability to adhere to KB epithelial cells. Both the type of medium (broth versus agar) and anaerobicity influenced adherence levels and cell surface characteristics. Optimal adherence was observed with all three strains after growth of the bacterial cells in broth under anaerobic conditions, a condition which was associated with extracellular microvesicles. Adherence of SUNY 75(S) also was correlated with extracellular amorphous material, whereas adherence of SUNY 465 was also associated with fimbriation which accompanied a smooth to rough phenotype shift. The relationship between adherence and extracellular vesicles, extracellular amorphous material, and fimbriation suggests that all of these components may function in A. actinomycetemcomitans adherence to epithelial cells. The phenotype shift observed in SUNY 465 cells is further evidence that A. actinomycetemcomitans SUNY 465 is predisposed to variant shifts which are associated with changes in adherence and invasion properties.
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PMCID: PMC186205  PMID: 8112865
4.  Surface properties of Streptococcus sanguis FW213 mutants nonadherent to saliva-coated hydroxyapatite. 
Infection and Immunity  1985;47(3):752-759.
Seventeen mutants of Streptococcus sanguis FW213 nonadherent to saliva-coated spheroidal hydroxyapatite were isolated after mutagenesis with ethyl methanesulfonate, nitrosoguanidine, nitrous acid, hydroxylamine, or 2-aminopurine. Enrichment for nonadherent mutants was accomplished by successive adsorptions of the adherent strains to saliva-coated hydroxyapatite. After enrichment, variant colonial morphology on tryptic agar was used as a screening technique for selection of nonadherent mutants, with loss of colonial opacity frequently associated with loss of adherence ability. These mutants were further characterized for additional surface properties, including twitching motility, saliva-induced aggregation, coaggregation with Actinomyces species, surface hydrophobicity, and presence of fimbriae. Results from these assays indicated that the nonadherent mutants fell into six phenotypic groups. A correlation between the loss of adherence ability, a decrease in cell fimbriation, and a decrease in surface hydrophobicity is apparent.
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PMCID: PMC261379  PMID: 2857684
5.  Identification of an immunoglobulin Fc receptor of Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1994;62(10):4500-4505.
Actinobacillus actinomycetemcomitans expresses proteins that bind to the Fc portion of immunoglobulins. The immunoglobulin Fc receptors on the surface of A. actinomycetemcomitans were detected by the binding of biotinylated human or murine Fc molecules to strain SUNY 465 adsorbed to the bottom of microtiter wells. Biotinylated Fc binding was inhibited by unlabeled Fc molecules and human plasma. Fc receptors were identified by the binding of biotinylated Fc molecules to bacterial membrane proteins separated by polyacrylamide gel electrophoresis and transferred to nitrocellulose. Multiple bands were identified, and the major Fc-binding protein was determined to be a heat-modifiable protein. This protein migrated with approximate molecular weights of 25,000 and 32,000 (unheated and heated, respectively). Amino-terminal sequence analysis of this protein revealed a sequence identical to the heat-modifiable protein described for A. actinomycetemcomitans ATCC 43718. This protein sequence exhibits significant homology with the N termini of outer membrane protein A (OmpA) of Escherichia coli and related OmpA-like proteins from other gram-negative bacteria.
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PMCID: PMC303136  PMID: 7927715
6.  Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line. 
Infection and Immunity  1994;62(9):3672-3678.
Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added. Variation in the level of adhesion was noted among strains of A. actinomycetemcomitans. Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M. Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells. Pretreatment of the KB cells with saliva did not inhibit adhesion. Protease treatment of A. actinomycetemcomitans reduced adhesion of the bacteria to KB cells. The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells.
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PMCID: PMC303017  PMID: 8063383
7.  Evidence that extracellular components function in adherence of Actinobacillus actinomycetemcomitans to epithelial cells. 
Infection and Immunity  1993;61(11):4933-4936.
Extracellular microvesicles and a highly proteinaceous polymer associated with a leukotoxin-producing strain, Actinobacillus actinomycetemcomitans SUNY 75, were shown to increase adherence of other weakly adherent A. actinomycetemcomitans strains to KB epithelial cells.
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PMCID: PMC281260  PMID: 8406899
8.  Evidence for invasion of a human oral cell line by Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1991;59(8):2719-2726.
Actinobacillus actinomycetemcomitans, an oral bacterial species associated with periodontal disease, was found to invade human cell lines. Invasion was demonstrated by recovery of viable organisms from gentamicin-treated KB cell monolayers and by light and electron microscopy. Internalization occurred through a cytochalasin D-sensitive process. Invasion efficiencies of some A. actinomycetemcomitans strains were comparable to those of invasive members of the family Enterobacteriaceae. Differences in invasiveness were correlated with bacterial colonial morphology. Smooth variants invaded more proficiently than rough variants. A. actinomycetemcomitans can undergo a smooth-to-rough colonial morphology shift which results in the loss of invasiveness. Coordinated regulation of genes involved in the rough-to-smooth phenotypic transitions may play a role in the episodic nature of periodontal disease.
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PMCID: PMC258078  PMID: 1855989
9.  Expression of Streptococcus sanguis antigens in Escherichia coli: cloning of a structural gene for adhesion fimbriae. 
Infection and Immunity  1987;55(1):123-128.
Chromosomal DNA from Streptococcus sanguis FW213 was partially digested with EcoRI and ligated into the positive-selection cloning vector pOP203(A2+). The ligation mixture was used to transform Escherichia coli K-12, and 4,500 transformants were examined. The tetracycline-resistant colonies had inserts averaging 3.2 kilobases. The entire colony bank was screened by colony immunoassay with polyclonal rabbit serum raised against S. sanguis FW213 whole cells. Thirty recombinant colonies produced stable positive reactions of various intensities, indicating that S. sanguis antigens could be expressed in E. coli. Restriction endonuclease digestion of these clones suggested that 26 of the clones were unique. Only two clones, VT616 and VT618, gave positive reactions with fimbria-specific antisera. That the gene coding for the antigen was located on the plasmid was confirmed by demonstrating that the presence of the plasmid was linked to antigen production. Western immunoblot analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels showed that both clones produced a fimbrial peptide of Mr 30,000. The two recombinant plasmids were shown by Southern analysis and restriction mapping to contain the same 6-kilobase EcoRI fragment inserted in opposite orientations. Southern hybridization confirmed that this fragment is present in S. sanguis genomic DNA. The Mr 30,000 protein gene was expressed in both orientations, suggesting that the fimbrial promoter is located on the 6-kilobase fragment. These results show that at least one streptococcal fimbrial gene can be cloned and expressed in E. coli.
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PMCID: PMC260289  PMID: 2878882
10.  Whole-bacterial cell enzyme-linked immunosorbent assay for Streptococcus sanguis fimbrial antigens. 
Journal of Clinical Microbiology  1982;16(1):141-144.
A whole-bacterial cell enzyme-linked immunosorbent assay (bactELISA) was developed for detecting fimbrial antigens on Streptococcus sanguis. In this assay, S. sanguis cells were directly adhered to polystyrene or polyvinyl via drying. Use of the assay indicated that consistently high and uniform optical densities could be obtained from well to well. In addition, radioactive assaying indicated increased adsorption to the polystyrene wells over polyvinyl, suggesting that polystyrene may prove superior in the gram-positive bactELISA. Use of the bactELISA may prove valuable to both the clinical and research laboratory involved in the study of bacterial cell surface components or in the evaluation of antisera directed against bacterial antigens, which are difficult to prepare as purified derivatives.
PMCID: PMC272310  PMID: 6125528

Results 1-10 (10)