Porphyromonas gingivalis mediates the shedding and proteolysis of complement regulatory protein CD46 expressed by oral epithelial cells.

INTRODUCTION: Human cells express membrane-bound complement regulatory proteins to prevent complement-mediated autologous tissue damage. In this study, we hypothesized that Porphyromonas gingivalis, the major etiological agent of chronic periodontitis, causes the shedding or proteolysis of the complement regulatory protein CD46 expressed by oral epithelial cells. METHODS: Oral epithelial cells were treated with a culture of P. gingivalis before measurement of membrane-bound and shed CD46 by enzyme-linked immunosorbent assay (ELISA). The effect of soluble recombinant CD46 on secretion of interleukin-8 (IL-8) by epithelial cells was evaluated by ELISA. The susceptibility of soluble recombinant CD46 to proteolytic degradation by cells and purified Lys-gingipain of P. gingivalis was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/western immunoblotting analysis. RESULTS: Oral epithelial cells treated with a culture of P. gingivalis showed a lower reactivity with antibodies directed to CD46. ELISA revealed that such a treatment resulted in increased amounts of CD46 in the conditioned media suggesting that P. gingivalis caused the shedding of membrane-anchored CD46. Stimulation of epithelial cells with soluble recombinant CD46 induced IL-8 secretion in a dose-dependent manner. Whole cells and purified Lys-gingipain of P. gingivalis degraded recombinant CD46 in a dose-dependent manner. CONCLUSION: This study showed the ability of P. gingivalis to induce the shedding/ proteolysis of CD46 from the surface of oral epithelial cells. This may render host cells susceptible to the complement system and contribute to tissue damage and the inflammatory process in periodontitis.

Potential oral health benefits of cranberry.

In the past decade, cranberry extracts have been attracting ever-growing attention by dental researchers. The potential benefits of cranberry components in reducing oral diseases, including dental caries and periodontitis, are discussed in this review. A non-dialysable cranberry fraction enriched in high molecular weight polyphenols has very promising properties with respect to cariogenic and periodontopathogenic bacteria, as well as to the host inflammatory response and enzymes that degrade the extracellular matrix. Cranberry components are potential anti-caries agents since they inhibit acid production, attachment, and biofilm formation by Streptococcus mutans. Glucan-binding proteins, extracellular enzymes, carbohydrate production, and bacterial hydrophobicity, are all affected by cranberry components. Regarding periodontal diseases, the same cranberry fraction inhibits host inflammatory responses, production, and activity of enzymes that cause the destruction of the extracellular matrix, biofilm formation, and adherence of Porphyromonas gingivalis, and proteolytic activities and coaggregation of periodontopathogens. The above-listed effects suggest that cranberry components, especially those with high molecular weight, could serve as bioactive molecules for the prevention and/or treatment of oral diseases.

Hemoglobin and LPS act in synergy to amplify the inflammatory response.

Vascular disruption and bleeding during periodontitis likely increase the levels of hemoglobin in gingival crevicular fluid. The aim of this study was to investigate the effect of hemoglobin on the inflammatory responses of human macrophages stimulated with lipopolysaccharides (LPS) isolated from periodontopathogens. The production of interleukin-1 beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) by macrophages following challenges with Porphyromonas gingivalis and Fusobacterium nucleatum LPS in the presence or absence of human hemoglobin was analyzed by ELISA. The effect of hemoglobin on LPS-binding to macrophages was evaluated with (3)H-LPS. Hemoglobin and LPS from periodontopathogens acted in synergy to stimulate the production of high levels of IL-1beta, IL-6, IL-8, and TNF-alpha by macrophages. Hemoglobin also enhanced LPS-binding to macrophages. This study suggests that hemoglobin contributes to increases in the levels of pro-inflammatory mediators in periodontal sites by acting in synergy with LPS from periodontopathogens, thus favoring the progression of periodontitis.

Inhibition of host extracellular matrix destructive enzyme production and activity by a high-molecular-weight cranberry fraction.

BACKGROUND AND OBJECTIVE: Periodontal diseases are a group of inflammatory disorders that are initiated by specific gram-negative bacteria and lead to connective tissue destruction. Proteolytic enzymes, including matrix metalloproteinases (MMPs) and elastase, produced by resident and inflammatory cells in response to periodontopathogens and their products, play a major role in gingival tissue destruction. The aim of this study was to investigate the effect of a high-molecular-weight fraction prepared from cranberry juice concentrate on MMP-3, MMP-9 and elastase activities, as well as on MMP production by human cells stimulated with lipopolysaccharide of Actinobacillus actinomycetemcomitans. MATERIAL AND METHODS: MMP-3 and MMP-9 production by gingival fibroblasts and macrophages treated with the cranberry fraction and then stimulated with lipopolysaccharide was measured by enzyme-linked immunosorbent assay. MMP-3, MMP-9 and elastase activities in the presence of the cranberry fraction were evaluated using colorimetric or fluorogenic substrates. The changes in expression and phosphorylation state of fibroblast intracellular signaling proteins induced by A. actinomycetemcomitans lipopolysaccharide and the cranberry fraction were characterized by antibody microarrays. RESULTS: The lipopolysaccharide-induced MMP-3 and MMP-9 responses of fibroblasts and macrophages were inhibited in a dose-dependent manner by the cranberry fraction. This fraction was found to inhibit fibroblast intracellular signaling proteins, a phenomenon that may lead to a down-regulation of activating protein-1 activity. MMP-3, MMP-9 and elastase activities were also efficiently inhibited by the cranberry fraction, even when it was used at low concentrations. CONCLUSION: These results suggest that cranberry compounds offer promising perspectives for the development of novel host-modulating strategies for an adjunctive treatment of periodontitis.

[Pathogenic potential of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, the red bacterial complex associated with periodontitis]. / Potentiel pathogénique de Porphyromonas gingivalis, Treponema denticola et Tannerella forsythia, le complexe bactérien rouge associé à la parodontite.

Periodontitis are mixed bacterial infections leading to destruction of tooth-supporting tissues, including periodontal ligament and alveolar bone. Among over 500 bacterial species living in the oral cavity, a bacterial complex named "red complex" and made of Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia has been strongly related to advanced periodontal lesions. While periodontopathogenic bacteria are the primary etiologic factor of periodontitis, tissue destruction essentially results from the host immune response to the bacterial challenge. Members of the red complex are Gram negative anaerobic bacteria expressing numerous virulence factors allowing bacteria to colonize the subgingival sites, to disturb the host defense system, to invade and destroy periodontal tissue as well as to promote the immunodestructive host response. This article reviews current knowledge of the pathogenic mechanisms of bacteria of the red complex leading to tissue and alveolar bone destruction observed during periodontitis.

Anti-inflammatory activity of a high-molecular-weight cranberry fraction on macrophages stimulated by lipopolysaccharides from periodontopathogens.

Periodontitis is a chronic inflammatory disease affecting oral tissues. The continuous, high production of cytokines by host cells triggered by periodontopathogens is thought to be responsible for the destruction of tooth-supporting tissues. Macrophages play a critical role in this host inflammatory response to periodontopathogens. The aim of this study was to investigate the effect of non-dialyzable material prepared from cranberry juice concentrate on the pro-inflammatory cytokine response of macrophages induced by lipopolysaccharides (LPS) from Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum subsp. nucleatum, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, and Escherichia coli. Interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and Regulated on Activation Normal T-cell Expressed and Secreted (RANTES) production by macrophages treated with the cranberry fraction prior to stimulation by LPS was evaluated by ELISA. Our results clearly indicate that the cranberry fraction was a potent inhibitor of the pro-inflammatory cytokine and chemokine responses induced by LPS. This suggests that cranberry constituents may offer perspectives for the development of a new therapeutic approach to the prevention and treatment of periodontitis.

Porphyromonas gingivalis-induced inflammatory mediator profile in an ex vivo human whole blood model.

Periodontitis is characterized by an accumulation of inflammatory cells in periodontal tissue and subgingival sites. Leukocytes play a major role in the host response to Porphyromonas gingivalis, a major aetiological agent of chronic periodontitis. Secretion of high levels of inflammatory mediators, including cytokines and prostaglandins, by leucocytes is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of an ex vivo whole blood model to P. gingivalis stimulation. The production of interleukin-1 beta (IL-1beta), IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, tumour necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), IFN-gamma-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), Regulated on Activation Normal T cell Expressed and Secreted (RANTES) and prostaglandin E2 (PGE2) were quantified by enzyme-linked immunosorbent assays. P. gingivalis induced the secretion of the pro-inflammatory cytokines IL-1beta, TNF-alpha, IL-6 and IFN-gamma, the chemokines IL-8, RANTES and MCP-1 and the inflammatory mediator PGE2 in an ex vivo human whole blood model. The secretion levels were dependent on the strain and the infectious dose used. While the mediator profiles were comparable between six healthy subjects, a high interindividual variability in the levels of secreted mediators was observed. This study supports the view that P. gingivalis, by inducing high levels of inflammatory mediators from a mixed leucocyte population, can contribute to the progression of periodontitis.

Streptococcus salivarius fimbriae are composed of a glycoprotein containing a repeated motif assembled into a filamentous nondissociable structure.

Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivarius fimbriae did not dissociate when they were incubated at 100 degrees C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 x 10(6) to 40 x 10(6) Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 microm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/phi, where X represents a modified amino acid residue and phi represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis and Streptococcus constellatus.

CD40 associates with the MHC class II molecules on human B cells.

The MHC class II and CD40 molecules are two major components of the immune system that are involved in cell-cell interactions and signal transduction. Data obtained in the course of the present investigation show that these two molecules are physically associated on the surface of various human B cell lines and on normal tonsilar B cells. The CD40 / MHC class II complexes were not detected on the germinal center B cell line Ramos. However, stimulation of these cells via CD40 or MHC class II triggered their association, suggesting that the formation of the complex is related to the activation status of the cells. The formation of these complexes did not alter the interaction of MHC class II molecules with one of their natural ligands, the staphylococcal enterotoxin A (SEA), as evidenced by the ability of SEA to bind MHC class II / CD40 complexes. Cross-linking of MHC class II or CD40 molecules leads to the association as well as the co-association of both molecules to the NP-49-insoluble cellular matrix. Such association allowed us to demonstrate that only a fraction of these molecules can be physically associated on the cell surface. Based on previous observations and those presented here, it is highly possible that the CD40 / MHC class II complexes may have an important role in signal(s) induced via both molecules and during T / B cells interactions.

Fimbriae and the hemagglutinating adhesin HA-Ag2 mediate adhesion of Porphyromonas gingivalis to epithelial cells.

The mechanisms by which Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is pathogenic for the periodontium remain largely hypothetical. Invasion of host tissues by P. gingivalis is believed to require adhesion of the bacterium to host cells. The aim of this study was to use monoclonal antibodies (MAbs) to characterize the bacterial cell surface component(s) acting as a ligand binding to a receptor on epithelial cells. Surface antigens of P. gingivalis ATCC 33277 were obtained as a glass bead-EDTA extract (GBE), and antiserum against the GBE was produced in rabbits. Epithelial cell membrane proteins (ECMP) were prepared from a homogenate of the SK-MES-1 cell line with Triton X-100. The antigen/ligand profile of GBE was resolved by crossed immunoaffinity electrophoresis by using ECMP in the first-dimension gel. The migration of one immunoprecipitate (IP) was retarded, indicating a ligand-receptor interaction between a surface antigen of P. gingivalis and a complementary binding site on the epithelial cell membrane. The corresponding IP in the GBE/anti-GBE immunoelectrophoresis profile was excised from replicate gels to immunize mice for production of MAbs specific for the bacterial ligand. Five MAbs were obtained and tested for reactivity with GBE in immunoblots and for inhibition of the interaction between GBE and ECMP. Immunoblots revealed polypeptides at 28, 42, 43, and 49 kDa. Inhibition tests were positive for all five MAbs. These results are conclusive evidence that the MAbs recognize functional epitopes involved in the adherence of P. gingivalis to epithelial cells and that the adhesins are likely associated with fimbriae and the hemagglutinating adhesin HA-Ag2.

Conservation of fimbriae and the hemagglutinating adhesin HA-Ag2 among Porphyromonas gingivalis strains and other anaerobic bacteria studied by epitope mapping analysis.

Monoclonal antibodies characterized as antifimbria and anti-HA-Ag2 were used in immunoblotting to examine the antigenic distribution of fimbriae and HA-Ag2 among a collection of human and animal Porphyromonas strains and human Prevotella and Bacteroides strains. The results showed that fimbrial and HA-Ag2 antigenic structures are peculiar to the species Porphyromonas gingivalis.

Detection of Bacteroides forsythus by immunomagnetic capture and a polymerase chain reaction-DNA probe assay.

The aim of this study was to combine immunomagnetic capture and a polymerase chain reaction (PCR) followed by hybridization with a DNA probe for the detection of Bacteroides forsythus. Magnetic beads were coated with the immunoglobulin G fraction of an antiserum specific for B. forsynthus. Aliquots were incubated with various concentrations of a suspension of B. forsythus or with a suspension containing 16 bacterial species, at a concentration of 10(10) cells/ml, spiked with dilutions of B. forsythus. Beads with bound bacteria were boiled, and the target DNA in the supernatant was amplified to generate a 392-bp PCR fragment specific for B. forsythus. The amplified product was detected by dot-blot hybridization with a digoxigenin-labeled 392-bp probe. The detection limit was determined to be 10 cells/ml using immunocapture on a suspension of B. forsythus and 100 on spiked bacterial suspensions. Subgingival plaque samples were obtained from 39 Bolivian individuals with poor oral hygiene. Each sample was analyzed by the above procedure and by immunofluorescence. The overall prevalence of individuals harboring B. forsythus was 62% by immunofluorescence and 82% by PCR-DNA probe assay. The immunocapture, PCR. DNA-probe procedure should be useful for the detection of B. forsythus, particularly in false-negative samples obtained by less sensitive techniques.

Selection and phenotypic characterization of nonhemagglutinating mutants of Porphyromonas gingivalis.

To further investigate the relationship between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis, three spontaneous mutants of the type strain ATCC 33277 were selected by a hemadsorption procedure. They were characterized for hemagglutination, trypsin-like and lectin-binding activities, and hydrophobicity and for the presence of fimbriae. The presence of the 42-kDa (the fimbrilin subunit) and the 43- and 49-kDa (the HA-Ag2 components) polypeptides was investigated by immunoblotting using polyclonal and monoclonal antibodies directed to fimbriae and to the hemagglutinating adhesin HA-Ag2. Cells from two of the three mutants (M1 and M2) exhibited no or little hemagglutination activity and very low trypsin-like activity and did not show the 43- and 49-kDa polypeptides. Abnormal fimbriation in M1 was deduced from the following observations of cells grown for 18 h: absence of the 42-kDa polypeptide and of a 14-kDa polypeptide and no fimbriae visible on electron micrographs. While the cells of mutant M2, irrespective of the age of the culture, were found to lack the 43- and 49-kDa polypeptides and hemagglutination activity, the supernatants of cultures grown for 72 h had high hemagglutination and trypsin-like activities and revealed the presence of the 42-, 43-, and 49-kDa polypeptides. This suggests that M2 may be missing some molecules which anchor the components to the cell surface. Mutant M3 showed levels of activities similar to those of the parental strain but lacked the 43-kDa polypeptide. Other pleiotropic effects observed for the mutants included loss of dark pigmentation and lower hydrophobicity. The data from this study fuel an emerging consensus whereby fimbriation, hemagglutination, and proteolytic activities, as well as other functions in P. gingivalis, are intricate.

Antigenic, structural, and functional relationships between fimbriae and the hemagglutinating adhesin HA-Ag2 of Porphyromonas gingivalis.

While the adhesive properties of Porphyromonas gingivalis are known to allow colonization of the subgingival tissues, the roles of fimbriae and adhesin molecules in hemagglutination remain unclear. The purpose of this study was to analyze the antigenic, structural, and functional relationships of these two components. Five populations of monoclonal antibodies were produced against (i) the hemagglutinating adhesin HA-Ag2 resolved by crossed immunoelectrophoresis (CIE), (ii) native fimbriae, and (iii) each of the three immunoprecipitates, Ag8a, Ag8b, and Ag8c, that define fimbriae by CIE. The tests used for characterization of the monoclonal antibodies included immunoblot reactivity, inhibition of hemagglutination, capacity to dissociate immunoprecipitates by CIE, localization of recognized epitopes by immunoelectron microscopy, and epitope mapping by competition enzyme-linked immunosorbent assay. The results from the different immunochemical tests clearly showed a close antigenic relationship between fimbriae and the hemagglutinating adhesin HA-Ag2. We were able to establish that the epitopic domain H1 of HA-Ag2 is hemagglutinin specific and that domain F2 is fimbria specific. Our data indicate that the polymeric structural unit of fimbriae must be complexed to HA-Ag2, the adhesin, to confer hemagglutination activity to the bacterial cells.

Molecular size variation of the hemagglutinating adhesin HA-Ag2, a common antigen of Bacteroides gingivalis.

The array of Bacteroides gingivalis W83 antigens revealed by crossed immunoelectrophoresis includes one antigen that is associated with an erythrocyte-binding capacity, termed the hemagglutinating adhesin HA-Ag2. This antigen was excised from crossed-immunoelectrophoresis plates to produce two polyclonal antisera, VL 011 and WL 303, whose restricted specificity for HA-Ag2 was assessed using crossed immunoelectrophoresis, crossed immunoelectrophoresis with an intermediate gel, and crossed immunoaffinoelectrophoresis. Both antisera, when used to probe blots of an EDTA cell surface extract of B. gingivalis W83, reacted with two bands, at 33 and 38 kDa, which were also detected by a monoclonal antibody (Naito et al. 1985. Infect. Immun. 50: 231-235), specific for a hemagglutinin of B. gingivalis. Antiserum WL 303 was used to examined by immunoblotting the distribution of HA-Ag2 among a variety of human and animal strains of B. gingivalis. All human strains tested showed two major bands at 33 and 38 kDa in the EDTA cell surface extract, and at 43 and 49 kDa in outer membrane preparations. Only one band, at 29 kDa, was detected in EDTA cell surface extracts from the animal strains, while the outer membrane preparation of a single strain showed a positive reaction. We concluded that HA-Ag2 is an antigen common to human and animal strains of B. gingivalis and that its subunits may show heterogeneity in apparent molecular mass.