Gene expression and phenotypic traits of Aggregatibacter actinomycetemcomitans in response to environmental changes.

BACKGROUND AND OBJECTIVE: Periodontopathogens experience several challenges in the oral cavity that may influence their transcription profile and resulting phenotype. This study evaluated the effect of environmental changes on phenotype and gene expression in a serotype b Aggregatibacter actinomycetemcomitans isolate. MATERIAL AND METHODS: Cultures in early exponential phase and at the start of stationary growth phase in microaerophilic and anaerobic atmospheres were evaluated. Cell hydrophobic properties were measured by adherence to n-hexadecane; in addition, adhesion to, and the ability to invade, KB cells was evaluated. Relative transcription of 12 virulence-associated genes was determined by real-time reverse transcritption quantitative PCR. RESULTS: The culture conditions tested in this study were found to influence the phenotypic and genotypic traits of A. actinomycetemcomitans. Cells cultured in microaerophilic conditions were the most hydrophobic, reached the highest adhesion efficiency and showed up-regulation of omp100 (which encodes an adhesion) and pga (related to polysaccharide synthesis). Cells grown anaerobically were more invasive to epithelial cells and showed up-regulation of genes involved in host-cell invasion or apoptosis induction (such as apaH, omp29, cagE and cdtB) and in adhesion to extracellular matrix protein (emaA). CONCLUSION: Environmental conditions of different oral habitats may influence the expression of factors involved in the binding of A. actinomycetemcomitans to host tissues and the damage resulting thereby, and thus should be considered in in-vitro studies assessing its pathogenic potential.

[Comparative study on the ability of adhesion and invasion of different fimA genetypes of Porphyromonas gingivalis to oral epithelial cells].

OBJECTIVE: To compare the ability of adhesion and invasion to epithelial cells by Porphyromonas gingivalis (Pg) strains with different fimA separated from Chinese. METHODS: Cultured method and antibiotic protection method were used to determine the adhesive and invasive ability of Pg with different fimA genetypes. The adhesion was observed by scanning electron microscope. RESULTS: All the strains adhered and invaded to KB cells, and the adhesion rate ranged from 0.523% to 37.125% and invasive rate from 0.017% to 3.750%.The adhesive and invasive ability among different fimA genotypes showed no significant difference (P > 0.05). CONCLUSIONS: There is no significant correlation between fimA genotype and ability in adhesion and invasion to KB cells.

Desulfovibrio spp. survive within KB cells and modulate inflammatory responses.

Desulfovibrio are sulfate-reducing anaerobic gram-negative rods that have been proposed as potential periodontopathogens. We investigated the capacity of Desulfovibrio to invade epithelial cells and induce cytokine secretion from these cells. Desulfovibrio strains were co-cultured with KB cells and counts of intracellular bacteria evaluated up to 3 days after infection. Desulfovibrio desulfuricans and Desulfovibrio fairfieldensis were able to survive within epithelial cells. Intracytoplasmic location of both bacterial species was confirmed by confocal laser scanning microscopy and transmission electron microscopy. Invasion was sensitive to nocodazole, an inhibitor of microtubule polymerization, but not to cytochalasin D, a microfilament inhibitor, suggesting that microtubule rearrangements were involved in the internalization of Desulfovibrio strains by KB cells. Infection by Desulfovibrio resulted in increased production of IL-6 and IL-8 by KB cells. The ability of D. desulfuricans and D. fairfieldensis to survive within oral epithelial cells and to modulate the epithelial immune response may contribute to the initiation and progression of periodontal diseases.

Influence of serum on interaction of Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 with an epithelial cell line.

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the influence of serum on the interaction of periodontal pathogens with epithelial cells using an epithelial cell line (KB cells). This is important because serum is a key component of gingival crevicular fluid and may influence inflammatory responses in epithelial cells exposed to periodontal pathogens. MATERIAL AND METHODS: Porphyromonas gingivalis ATCC 33277 and Aggregatibacter actinomycetemcomitans Y4 were co-cultured with KB cells either with or without the addition of up to 10% human serum or 50 mg/mL human serum albumin. The numbers of free-floating, adherent and intracellular bacteria were determined up to 18 h after exposure of the epithelial cells to the pathogens. Additionally, the concentrations of interleukin (IL)-6 and IL-8 produced by the epithelial cells in response to exposure to the bacteria were determined. RESULTS: Serum and human serum albumin reduced the number of internalized A. actinomycetemcomitans Y4 organisms in the epithelial cells, increased the levels of IL-6 and IL-8 in the supernatants of infected cells (those with internalized A. actinomycetemcomitans) and influenced non-infected epithelial cells. Increased IL-6 and IL-8 concentrations were also detected in the supernatants of KB cells infected with P. gingivalis ATCC 33277. Interleukin-6 and IL-8 were detectable after addition of serum, probably as a result of inhibition of the activity of P. gingivalis cysteine proteinases by serum. CONCLUSION: Serum promotes the release of the cytokines IL-6 and IL-8 by epithelial cells. This mechanism is influenced by periodontal pathogens and may maintain clinical periodontal inflammation.

Flow cytometric analysis of adherence of Porphyromonas gingivalis to oral epithelial cells.

By using fluorescence microscopy, fluorescently labeled Porphyromonas gingivalis W50 was shown to adhere to oral epithelial (KB) cells as discrete cells or small cell aggregates, whereas P. gingivalis ATCC 33277 bound as large cell aggregates. Flow cytometric analysis showed that for P. gingivalis W50 there was a logarithmic relationship between the bacterial cell ratio (BCR), that is the number of bacterial cells to KB cells, and the percentage of KB cells with W50 cells attached. This percentage of KB cells with W50 attached reached a plateau of approximately 84% cells at a BCR of 500:1. In contrast, a quadratic relationship was observed between BCR and the percentage of KB cells with P. gingivalis ATCC 33277 attached, reaching a maximum of 47% at a BCR of 100:1 but decreasing to 7% at a BCR of 1,000:1. The lower binding of ATCC 33277 at high cell concentrations was attributed to autoaggregation. P. gingivalis W50 cells treated with an inhibitor (Nalpha-p-tosyl-L-lysine chloromethyl ketone [TLCK]) of its RgpA-Kgp proteinase-adhesin complex exhibited significantly reduced binding to KB cells than to untreated cells, suggesting a role for proteinase activity in binding to KB cells. Competitive inhibition with purified proteinase-active and TLCK-inactivated RgpA-Kgp complex significantly decreased the adherence of P. gingivalis W50 cells to KB cells. Furthermore, isogenic mutants of P. gingivalis W50 lacking the kgp gene product, but not the rgpA or rgpB gene products, exhibited significantly decreased adherence to KB cells compared to the wild type.

Surface properties of Lactobacilli isolated from healthy subject.

Lactobacilli were isolated from mouth, stomach and intestine of healthy subjects and identified by using standard bacteriological methods. The aim of the study was to determine the cell surface characteristics of Lactobacilli and to correlate them with ability to adhere to KB cell line. Our results revealed that all isolated Lactobacillus strains showed basic surface character and those with high hydrophobicity scores and auto-aggregation were highly adherent to KB cell line.

Porphyromonas gingivalis survives within KB cells and modulates inflammatory response.

BACKGROUND/AIMS: The purpose of the study was to investigate the intracellular survival of Porphyromonas gingivalis as a possible mechanism for maintaining periodontitis. METHODS: P. gingivalis strains, the strain ATCC 33277 and seven clinical isolates, were co-cultured with KB cells. The number of intracellular bacteria was determined up to 3 days after infection. In addition, the numbers of KB cells per well, the concentrations of the cytokines interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha) and the arginine-specific amidolytic activity were measured. The 16S rRNA of P. gingivalis and the mRNA expression of IL-1beta, IL-6, IL-8, TNF-alpha and rgpA were also determined. RESULTS: All the P. gingivalis strains studied were able to survive within KB cells. In contrast to the reduced values of colony-forming units at day 3, equal and higher levels of 16S rRNA were seen in comparison to day 0. Arginine-specific amidolytic activity declined in all samples during infection. Expression of mRNA for rgpA was not found after infection of KB cells by P. gingivalis strains. IL-8 was detectable in all samples 2 days after infection with P. gingivalis strains. Principal components analysis underlined a correlation between the arginine-specific amidolytic activity 1 h after infection and both the released IL-8 and the mRNA expression of IL-8. Associations were found between the cultivable numbers of intracellular P. gingivalis and the mRNAs of IL-1, IL-6 and TNF-alpha at the day of infection. CONCLUSION: The results indicate survival of P. gingivalis within epithelial cells, possibly in a non-cultivable stage. Invasion into cells modulates the virulence properties of P. gingivalis as well as the inflammatory response of the cells.

Inactivation of DNA adenine methyltransferase alters virulence factors in Actinobacillus actinomycetemcomitans.

DNA adenine methyltransferase (DAM) plays critical roles in diverse biological pathways in gram-negative bacteria, and specifically in regulating the expression of virulence genes in several organisms. Actinobacillus actinomycetemcomitans plays an important role in the pathogenesis of juvenile and adult periodontal disease, yet little is known about its mechanisms of gene regulation. DAM is shown here to directly or indirectly affect well-known A. actinomycetemcomitans virulence factors. A mutant A. actinomycetemcomitans strain lacking the dam gene was created by homologous recombination and shows normal growth phenotypes when grown exponentially. This mutant strain has four sixfold increased levels of extracellular leukotoxin, altered cellular levels of leukotoxin, and significant changes in bacterial invasion of KB oral epithelial cells. These results provide a basis for further characterization of regulatory mechanisms that control A. actinomycetemcomitans virulence.

Distinctive characteristics of transcriptional profiles from two epithelial cell lines upon interaction with Actinobacillus actinomycetemcomitans.

Transcriptional profiling and gene ontology analyses were performed to investigate the unique responses of two different epithelial cell lines to an Actinobacillus actinomycetemcomitans challenge. A total of 2867 genes were differentially regulated among all experimental conditions. The analysis of these 2867 genes revealed that the predominant specific response to infection in HeLa cells was associated with the regulation of enzyme activity, RNA metabolism, nucleoside and nucleic acid transport and protein modification. The predominant specific response in immortalized human gingival keratinocytes (IHGK) was associated with the regulation of angiogenesis, chemotaxis, transmembrane receptor protein tyrosine kinase signaling, cell differentiation, apoptosis and response to stress. Of particular interest, stress response genes were significantly - yet differently - affected in both cell lines. In HeLa cells, only three regulated genes impacted the response to stress, and the response to unfolded protein was the only term that passed the ontology filters. This strikingly contrasted with the profiles obtained for IHGK, in which 61 regulated genes impacted the response to stress and constituted an extensive network of cell responses to A. actinomycetemcomitans interaction (response to pathogens, oxidative stress, unfolded proteins, DNA damage, starvation and wounding). Hence, while extensive similarities were found in the transcriptional profiles of these two epithelial cell lines, significant differences were highlighted. These differences were predominantly found in pathways that are associated with host-pathogen interactions.

Expression of endothelin-1 in gingival epithelial cells.

OBJECTIVES AND BACKGROUND: Endothelin-1 (ET-1) is a 21-amino acid peptide with multifunctional regulation. ET-1 expresses in various cells during inflammation. The present study aimed to examine the ET-1 expression in oral epithelial cells after infection with the periodontal pathogen and to investigate the presence of ET-1 in human inflamed and uninflamed gingival tissues. MATERIALS AND METHODS: The KB cells were infected with Porphyromonas gingivalis and the expression level of ET-1 was examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The immunohistochemical analysis of ET-1 was performed in gingival tissues obtained from patients. In addition, the ET-1 mRNA expression in each tissue was also investigated by RT-PCR. RESULTS: The expression of ET-1 in KB cells was strongly induced by the P. gingivalis infection. On the other hand, the strong immunoreactivity for ET-1 was observed in the epithelium and vascular endothelial cells of the inflamed gingival tissue. Furthermore, the level of ET-1 mRNA was greater in the inflamed tissues. CONCLUSION: These results suggested that the expression level of ET-1 in gingival epithelial cells might be enhanced during the periodontal inflammation.

Epithelial cell surface sites involved in the polyvalent adherence of Porphyromonas gingivalis: a convincing role for neuraminic acid and glucuronic acid.

We investigated the target structures of the epithelial cells responsible for the attachment of Porphyromonas gingivalis by immunocytofluorimetry, enzyme-linked immunosorbent assay, and confocal microscopy. Integrins (beta1, beta3, and alphaV) and E-cadherin played no significant role. Carbohydrates (such as alpha-D-methylglucoside, L-fucose, D- and L-mannose, N-acetylglucosamine, and N-acetylgalactosamine) had little inhibitory effect on bacterial binding. Enzymatic treatments of the epithelial membranes and sugar competition studies showed that N-acetylneuraminic acid and glucuronic acid were involved in binding.

N-acetyl-D-galactosamine specific lectin of Eikenella corrodens induces intercellular adhesion molecule-1 (ICAM-1) production by human oral epithelial cells.

During the acute inflammatory response in periodontitis, gingival epithelial cells are considered to play important roles in the recruitment of inflammatory cells to the site of infection through the secretion of chemokines. However, little is known about the expression of molecules that are involved in the interaction between the epithelium and neutrophils following bacterial attachment. Earlier work reported that periodontopathogenic Eikenella corrodens strain 1,073 up-regulated the expression and secretion of chemokines such as interleukin-8 (IL-8) from KB cells (a human oral epithelial cell line derived from a human oral epidermoid carcinoma). To elucidate the mechanism of the transmigration of neutrophils through the epithelium, the present study investigated the expression of adhesion molecules on KB cells in response to E. corrodens attachment. Adhesion molecule gene expression was assessed by RT-PCR and adhesion proteins expressed on KB cell surfaces were determined by cell-based ELISA and FACS. In RT-PCR, ICAM-1 mRNA levels were significantly increased within 1 h in response to exposure to E. corrodens and continued to increase over the 12-h period of study. In ELISA, increased surface ICAM-1 expression was paralleled by increased ICAM-1 mRNA levels. Furthermore, the increases in ICAM-1 expression on epithelial cells infected with E. corrodens were observed to be due to the N-acetyl-D-galactosamine (GalNAc) specific bacterial lectin-like substance of E. corrodens (EcLS), which was one of the adhesins of E. corrodens. This is the first study to report that a bacterial lectin-like substance increased the expression of ICAM-1 on gingival epithelial cells.

Interaction of Porphyromonas gingivalis with KB cells: comparison of different clinical isolates.

The ability of different Porphyromonas gingivalis strains (15 clinical isolates and ATCC 33277) to attach to and invade KB cells, in relation to other properties such as release of interleukin (IL)-6 and IL-8, cytotoxicity, proteolytic activity and types of fimbriae genes present, was examined. A hierarchical cluster analysis based on adherence and internalization resulted in four groups. Eight of the 15 clinical isolates belonged to a cluster group whose adherence and internalization were about 10% those of the ATCC strain. A negative correlation between lysine-specific protease activity and adherence was found. In all cases the released concentrations of IL-6 and IL-8 were very low. Only one strain was found to be cytotoxic to KB cells. Principal components analysis demonstrated correlations between adherence, internalization and autoaggregation. Most strains had fimA type I and II, type I being associated with elastase-like activity. The ability of P. gingivalis to invade epithelial cells may be a key factor for maintaining periodontal disease.

Kinetics of KB and HEp-2 cell responses to an invasive, cytolethal distending toxin-producing strain of Actinobacillus actinomycetemcomitans.

The periodontal pathogen Actinobacillus actinomycetemcomitans produces cytolethal distending toxin (CDT), a complex multicomponent toxin that arrests the growth of many types of eukaryotic cell. The kinetics of the effects of CDT-containing extracts, from an invasive strain of this bacterium, were examined on epithelial-like cells routinely used in invasion studies. Both KB and HEp-2 cells were exquisitely sensitive to the effects of the CDT with TD50 of 30 and 300 pg of total bacterial protein, respectively. Initial cell morphology changes were relatively rapid, occurring within the first 13 h of exposure. CDT-treated KB cells increased in size to 4-5 times the size of untreated controls. Cytotoxicity was irreversible when attached cells were incubated, for a minimum of 120 min, with nanogram quantities of CDT-containing extract. As cultures aged, the cells became more resistant to the effects of the CDT-containing extracts. These findings have important implications for understanding the ability of A. actinomycetemcomitans to invade and multiply in epithelial cells.

Soluble products from Eikenella corrodens stimulate oral epithelial cells to induce inflammatory mediators.

In the inflammatory response elicited by bacterial colonization in periodontal pockets, pocket epithelial cells not only serve as a barrier to isolate the pocket microenvironment from external stimuli but also regulate the functions of neighboring cells including fibroblasts and inflammatory cells. To elucidate this mechanism, we characterized the effects of periodontopathic bacterium Eikenella corrodens 1073 components on the production of some inflammatory mediators in a human oral epithelial cell line (KB). In enzyme-linked immunosorbent assay (ELISA), the E. corrodens supernatant induced interleukin-6 (IL-6), IL-8 and prostaglandin E2 but not interferon-gamma (IFN-gamma) production by KB cells. After incubation with E. corrodens supernatant, KB cells showed a marked increase in the levels of IL-6, IL-8 and PG G/H synthase (cyclooxygenase)-2, but not IFN-gamma, gene expression by reverse-transcriptase polymerase chain reaction. All these E. corrodens products responsible for production of these inflammatory mediators resisted freezing and boiling and were present in a 10-kDa filtrate. These results imply that these soluble small-molecular-mass products from E. corrodens stimulate various inflammatory mediator productions by human oral epithelial cells and may play a role in the initiation of periodontal inflammation and subsequently perpetuate the inflammatory response during chronic infection.

Calprotectin expression inhibits bacterial binding to mucosal epithelial cells.

Squamous mucosal epithelial cells constitutively express calprotectin in the cytoplasm. To study how this antimicrobial protein complex confers epithelial resistance to invading bacteria, an epithelial cell line was stably transfected to express the calprotectin complex. Cells expressing calprotectin resist invasion by Listeria monocytogenes and Salmonella enterica serovar Typhimurium. Calprotectin expression was accompanied by altered actin organization, increased alpha3 integrin expression, and spreading cell morphology. In this study, we assessed whether calprotectin expression affects bacterial binding and uptake. Threefold-fewer Listeria organisms bound to the surfaces of calprotectin-expressing cells, and 10-fold fewer were localized intracellularly by immunofluorescence. Similarly, fewer Salmonella organisms bound to cells expressing calprotectin. Calprotectin-expressing and sham-transfected cells showed similar levels of expression of surface E-cadherin and intracellular adhesion molecule 1 (ICAM-1) by flow cytometry. Calprotectin-expressing transfectants expressed calprotectin on the cell surface as well as in the cytosol. In conclusion, two bacterial pathogens showed reduced binding to calprotectin-expressing epithelial cells. Calprotectin-expressing cells appeared to have internalized disproportionately fewer Listeria organisms, suggesting that reduced binding and translocation supplemented direct antimicrobial effects in calprotectin-expressing cells.

Actinobacillus actinomycetemcomitans may utilize either actin-dependent or actin-independent mechanisms of invasion.

Actinobacillus actinomycetemcomitans is an important pathogen implicated in juvenile and adult periodontal diseases. An important virulence factor of A. actinomycetemcomitans is the ability to invade human oral epithelial cells. A clinical isolate, A. actinomycetemcomitans SUNY 465, has previously been shown to enter epithelial cells by an actin-dependent mechanism. The internalized bacteria are surrounded by an actin halo upon entry. These data are consistent with the mode of entry associated with many enteric pathogens. We tested the effects of cytochalasin D, an inhibitor of the actin microfilament network, on bacterial entry to determine whether this mode of entry was common to other A. actinomycetemcomitans clinical isolates. Cytochalasin D was added prior to infection. A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065 exhibited enhanced ability to enter epithelial cells in the presence of cytochalasin D. Immunofluorescent labeling of bacteria and host cell actin confirmed that actin was not being mobilized by the entry of A. actinomycetemcomitans SUNY 523. Inhibitors of receptor-mediated endocytosis inhibited invasion of A. actinomycetemcomitans SUNY 523 and A. actinomycetemcomitans 4065. Microtubule effectors did not inhibit invasion of A. actinomycetemcomitans. A. actinomycetemcomitans SUNY 523, but not A. actinomycetemcomitans 4065, was deficient in exit from epithelial cells as determined by the absence of organisms in the assay medium. These data suggest that A. actinomycetemcomitans strains utilize at least two distinct mechanisms for entry into epithelial cells, and that A. actinomycetemcomitans SUNY 523 may be defective in exit and cell-to-cell spread.

Epithelial cell invasion by Actinobacillus actinomycetemcomitans strains from restriction fragment-length polymorphism groups associated with juvenile periodontitis or carrier status.

The epithelial cell invasiveness of Actinobacillus actinomycetemcomitans strains of different restriction fragment-length polymorphism (RFLP) groups associated with disease conversion and asymptomatic carrier status in localized juvenile periodontitis was examined. Twenty clinical isolates were studied for their ability to invade KB monolayers, using the quantitative gentamicin killing assay. Five isolates were found to be invasive, five were not invasive; and the other 10 did not invade better than an invasion negative control Haemophilus aphrophilus strain ATCC 19415. Using probe-specific DNA fingerprinting. 11 strains were assigned to RFLP group II (disease-associated); 4 to RFLP type XIII (carrier status associated); and the other to groups III, IV, V and VII. Eight isolates, all RFLP group II, were leukotoxin producers as determined by PCR amplification of the lkt promoter region. No correlation was found between invasiveness and RFLP group. Leukotoxin production was more associated with noninvasive than invasive strains.

Functional interactions within adenovirus E1A protein complexes.

The transforming potential of adenovirus E1A oncogene products derives largely from the formation of complexes with cellular proteins, including the p105Rb tumor suppressor and a related p107 species, p130 and p300 proteins, and cyclin A (p60cycA). Extensive quantitative analyses using E1A deletion mutants identified unique binding patterns for each of these polypeptides within the amino terminus and conserved regions 1 and 2 (CR1 and CR2) of E1A proteins. A novel protein, termed p400, was found by peptide mapping to be related to p300, and, like p300, to require the E1A amino terminus and a portion of CR1 for binding. p130 was shown to be related to p107, and like p107, to associate with p60cycA. p107, p130 and p105Rb all interacted primarily with CR2, however, sequences within CR1 and the amino terminus were capable of weak interactions and appeared to function cooperatively with CR2 to bind these proteins. Protein kinase activity present in E1A complexes probably derives at least in part from p60cycA-linked p33cdk2 associated with p107 and p130. In vitro phosphorylation of complexes purified by immunoprecipitation resulted in labeling of several proteins. p60cycA was phosphorylated to about the same extent in cyclin A complexes prepared from either AD5- or mock-infected KB cells, however, that of p130 and p107 was dramatically higher in p60cycA complexes from infected cells. p300 was also phosphorylated in complexes prepared using E1A-specific antibodies. Thus one role of E1A proteins in signal transduction and regulation of the cell cycle may be to control the biological activity of p107, p130 and p300 by enhancing their phosphorylation through complex formation.

Porphyromonas gingivalis invades human pocket epithelium in vitro.

The present study examined the adhesive and invasive potential of Porphyromonas gingivalis interacting with human pocket epithelium in vitro. Pocket epithelial tissue, obtained during periodontal surgery of patients with advanced periodontal disease, generated a stratified epithelium in culture. P. gingivalis strains W50 and FDC 381 (laboratory strains), OMGS 712, 1439, 1738, 1739 and 1743 (clinical isolates) as well as Escherichia coli strain HB101 (non-adhering control) were tested with respect to epithelial adhesion and invasion. Adhesion was quantitated by scintillation spectrometry after incubation of radiolabeled bacteria with epithelial cells. The invasive ability of P. gingivalis was measured by means of an antibiotic protection assay. The epithelial multilayers were infected with the test and control strains and subsequently incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). The number of internalized bacteria surviving the antibiotic treatment was assessed after plating lyzed epithelial cells on culture media. All tested P. gingivalis strains adhered to and entered pocket epithelial cells. However, considerable variation in their adhesive and invasive potential was observed. E. coli strain HB101 did not adhere or invade. Transmission electron microscopy revealed that internalization of P. gingivalis was preceded by formation of microvilli and coated pits on the epithelial cell surfaces. Intracellular bacteria were most frequently surrounded by endosomal membranes; however, bacteria devoid of such membranes were also seen. Release of outer membrane vesicles (blebs) by internalized P. gingivalis was observed. These results support and extend previous work from this laboratory which demonstrated invasion of a human oral epithelial cell-line (KB) by P. gingivalis.