Interactions between Periodontal Bacteria and Human Oral Epithelial Cells: Fusobacterium nucleatum Adheres to and Invades Epithelial Cells

ABSTRACT Bacteria are causative agents of periodontal diseases. Interactions between oral bacteria and gingival epithelial cells are essential aspects of periodontal infections. Using an in vitro tissue culture model, a selected group of gram-negative anaerobic bacteria frequently associated with periodontal diseases, includingBacteroides forsythus, Campylobacter curvus,Eikenella corrodens, Fusobacterium nucleatum,Porphyromonas gingivalis, and Prevotella intermedia, were examined for their ability to adhere to and invade primary cultures of human gingival epithelial cells (HGEC). The effects of these bacteria on the production of interleukin-8 (IL-8), a proinflammatory chemokine, were also measured. These studies provided an initial demonstration that F. nucleatum adhered to and invaded HGEC and that this was accompanied by high levels of IL-8 secretion from the epithelial cells. The attachment and invasion characteristics of F. nucleatumwere also tested using KB cells, an oral epithelial cell line. The invasion was verified by transmission electron microscopy and with metabolic inhibitors. Invasion appeared to occur via a “zipping” mechanism and required the involvement of actins, microtubules, signal transduction, protein synthesis, and energy metabolism of the epithelial cell, as well as protein synthesis by F. nucleatum. A spontaneous mutant, lam, of F. nucleatum, isolated as defective in autoagglutination, was unable to attach to or invade HGEC or KB cells, further indicating the requirement of bacterial components in these processes. Sugar inhibition assays indicated that lectin-like interactions were involved in the attachment of F. nucleatum to KB cells. Investigation of these new virulence phenotypes should improve our understanding of the role of F. nucleatum in periodontal infections.

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The Influence of Oral Bacteria on Epithelial Cell MigrationIn Vitro

Mediators of Inflammation ◽

10.1155/2013/154532 ◽

2013 ◽

Vol 2013 ◽

pp. 1-6 ◽

Cited By ~ 17

Author(s):

Alexa M. G. A. Laheij ◽

Johannes J. de Soet ◽

Enno C. I. Veerman ◽

Jan G. M. Bolscher ◽

Cor van Loveren

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Oral Bacteria ◽

Bacterial Cells ◽

Oral Epithelial Cells ◽

Prevotella Nigrescens ◽

Hematopoetic Stem Cell ◽

Oral Epithelial ◽

Hematopoetic Stem Cell Transplantation

Oral ulcerations often arise as a side effect from chemo- and radiation therapy. In a previous clinical study,Porphyromonas gingivaliswas identified as a positive predictor for oral ulcerations after hematopoetic stem cell transplantation, possibly incriminatingP. gingivalisin delayed healing of the ulcerations. Therefore, it was tested whetherP. gingivalisand its secreted products could inhibit the migration of oral epithelial cells in anin vitroscratch assay. To compare, the oral bacteriaPrevotella nigrescens,Prevotella intermedia,Tannerella forsythia, andStreptococcus mitiswere included. A standardized scratch was made in a confluent layer of human oral epithelial cells. The epithelial cells were challenged with bacterial cells and with medium containing secretions of these bacteria. Closure of the scratch was measured after 17 h using a phase contrast microscope.P. gingivalis,P. nigrescens, and secretions ofP. gingivalisstrongly inhibited cell migration. A challenge with 1000 heat-killed bacteria versus 1 epithelial cell resulted in a relative closure of the scratch of 25% forP. gingivalisand 20% forP. nigrescens. Weaker inhibitory effects were found for the other bacteria. The results confirmed our hypothesis that the oral bacteria may be involved in delayed wound healing.

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Calprotectin Expression In Vitro by Oral Epithelial Cells Confers Resistance to Infection by Porphyromonas gingivalis

Infection and Immunity ◽

10.1128/iai.69.7.4242-4247.2001 ◽

2001 ◽

Vol 69 (7) ◽

pp. 4242-4247 ◽

Cited By ~ 98

Author(s):

K. Nisapakultorn ◽

K. F. Ross ◽

M. C. Herzberg

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Porphyromonas Gingivalis ◽

Calcium Binding ◽

Periodontal Diseases ◽

Epithelial Cell Line ◽

Transfected Cells ◽

Cell Rounding ◽

Oral Epithelial

ABSTRACT Calprotectin, an S100 calcium-binding protein with broad-spectrum antimicrobial activity in vitro, is expressed in neutrophils, monocytes, and gingival keratinocytes. In periodontitis, calprotectin appears upregulated and is detected at higher levels in gingival crevicular fluid and tissue specimens. How calprotectin contributes to the pathogenesis of periodontal diseases is unknown. To isolate the effects of calprotectin, a calprotectin-negative oral epithelial cell line was transfected with calprotectin genes to enable expression.Porphyromonas gingivalis was permitted to bind and invade transfected cells expressing calprotectin and sham transfectants. Rates of invasion into both cell lines were compared using the antibiotic protection assay. Transfected cells expressing calprotectin showed 40 to 50% fewer internalized P. gingivalis than sham transfectants. Similarly, binding to calprotectin expressing cells was reduced approximately twofold at all time points (15, 30, 45, and 60 min) as estimated by immunofluorescence analysis. Independent of invasion, however, prolonged exposure to P. gingivalisinduced epithelial cell rounding and detachment from the substratum. These morphological changes were delayed, however, in cells expressing calprotectin. Using P. gingivalis protease-deficient mutants, we found that Arg-gingipain and Lys-gingipain contributed to epithelial cell rounding and detachment. In conclusion, expression of calprotectin appears to protect epithelial cells in culture against binding and invasion by P. gingivalis. In addition, cells expressing calprotectin are more resistant to detachment mediated by Arg-gingipain and Lys-gingipain. In periodontal disease, calprotectin may augment both the barrier protection and innate immune functions of the gingival epithelium to promote resistance to P. gingivalis infection.

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Invasion of Human Oral Epithelial Cells byPrevotella intermedia

Infection and Immunity ◽

10.1128/iai.66.12.6054-6057.1998 ◽

1998 ◽

Vol 66 (12) ◽

pp. 6054-6057 ◽

Cited By ~ 70

Author(s):

Brian R. Dorn ◽

K.-P. Leung ◽

Ann Progulske-Fox

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Cell Line ◽

Clinical Isolate ◽

Oral Bacteria ◽

Epithelial Cell Line ◽

Oral Epithelial Cells ◽

Oral Epithelial Cell ◽

Type C ◽

Oral Epithelial

ABSTRACT Invasion of oral epithelial cells by pathogenic oral bacteria may represent an important virulence factor in the progression of periodontal disease. Here we report that a clinical isolate ofPrevotella intermedia, strain 17, was found to invade a human oral epithelial cell line (KB), whereas P. intermedia 27, another clinical isolate, and P. intermedia 25611, the type strain, were not found to invade the cell line. Invasion was quantified by the recovery of viable bacteria following a standard antibiotic protection assay and observed by electron microscopy. Cytochalasin D, cycloheximide, monodansylcadaverine, and low temperature (4°C) inhibited the internalization of P. intermedia 17. Antibodies raised against P. intermedia type C fimbriae and against whole cells inhibited invasion, but the anti-type-C-fimbria antibody inhibited invasion to a greater extent than the anti-whole-cell antibody. This work provides evidence that at least one strain ofP. intermedia can invade an oral epithelial cell line and that the type C fimbriae and a cytoskeletal rearrangement are required for this invasion.

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Fusobacterium nucleatum Transports Noninvasive Streptococcus cristatus into Human Epithelial Cells

Infection and Immunity ◽

10.1128/iai.74.1.654-662.2006 ◽

2006 ◽

Vol 74 (1) ◽

pp. 654-662 ◽

Cited By ~ 64

Author(s):

Andrew M. Edwards ◽

Tracy J. Grossman ◽

Joel D. Rudney

Keyword(s):

Epithelial Cells ◽

Cell Attachment ◽

Polyclonal Antibodies ◽

Fusobacterium Nucleatum ◽

Oral Bacteria ◽

Host Cells ◽

Oral Streptococci ◽

Kb Cells ◽

Actinomyces Naeslundii ◽

Adhesive Interactions

ABSTRACT Analysis of human buccal epithelial cells frequently reveals an intracellular polymicrobial consortium of bacteria. Although several oral bacteria have been demonstrated to invade cultured epithelial cells, several others appear unable to internalize. We hypothesized that normally noninvasive bacteria may gain entry into epithelial cells via adhesion to invasive bacteria. Fusobacterium nucleatum is capable of binding to and invading oral epithelial cells. By contrast, Streptococcus cristatus binds weakly to host cells and is not internalized. F. nucleatum and S. cristatus coaggregate strongly via an arginine-sensitive interaction. Coincubation of KB or TERT-2 epithelial cells with equal numbers of F. nucleatum and S. cristatus bacteria led to significantly increased numbers of adherent and internalized streptococci. F. nucleatum also promoted invasion of KB cells by other oral streptococci and Actinomyces naeslundii. Dissection of fusobacterial or streptococcal adhesive interactions by using sugars, amino acids, or antibodies demonstrated that this phenomenon is due to direct attachment of S. cristatus to adherent and invading F. nucleatum. Inhibition of F. nucleatum host cell attachment and invasion with galactose, or fusobacterial-streptococcal coaggregation by the arginine homologue l-canavanine, abrogated the increased S. cristatus adhesion to, and invasion of, host cells. In addition, polyclonal antibodies to F. nucleatum, which inhibited fusobacterial attachment to both KB cells and S. cristatus, significantly decreased invasion by both species. Similar decreases were obtained when epithelial cells were pretreated with cytochalasin D, staurosporine, or cycloheximide. These studies indicate that F. nucleatum may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly.

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A cocoa (Theobroma cacao L.) extract impairs the growth, virulence properties, and inflammatory potential of Fusobacterium nucleatum and improves oral epithelial barrier function

PLoS ONE ◽

10.1371/journal.pone.0252029 ◽

2021 ◽

Vol 16 (5) ◽

pp. e0252029

Author(s):

Amel Ben Lagha ◽

Patricia Maquera Huacho ◽

Daniel Grenier

Keyword(s):

Epithelial Cells ◽

Barrier Function ◽

Theobroma Cacao ◽

Biofilm Formation ◽

Periodontal Diseases ◽

Fusobacterium Nucleatum ◽

Epithelial Barrier ◽

Epithelial Barrier Function ◽

Oral Epithelial Cells ◽

Oral Epithelial

Fusobacterium nucleatum is associated with many conditions and diseases, including periodontal diseases that affect tooth-supporting tissues. The aim of the present study was to investigate the effects of a cocoa extract (Theobroma cacao L.) on F. nucleatum with respect to growth, biofilm formation, adherence, and hydrogen sulfide (H2S) production. The anti-inflammatory properties and the effect on epithelial barrier function of the cocoa extract were also assessed. The cocoa extract, whose major phenolic compound is epicatechin, dose-dependently inhibited the growth, biofilm formation, adherence properties (basement membrane matrix, oral epithelial cells), and H2S production of F. nucleatum. It also decreased IL-6 and IL-8 production by F. nucleatum-stimulated oral epithelial cells and inhibited F. nucleatum-induced NF-κB activation in monocytes. Lastly, the cocoa extract enhanced the barrier function of an oral epithelial model by increasing the transepithelial electrical resistance. We provide evidence that the beneficial properties of an epicatechin-rich cocoa extract may be useful for preventing and/or treating periodontal diseases.

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Possible Roles of IL-33 in Periodontal Diseases: Porphyromonas gingivalis Induced IL-33 in Human Gingival Epithelial Cells

Interface Oral Health Science 2014 ◽

10.1007/978-4-431-55192-8_25 ◽

2015 ◽

pp. 293-303

Author(s):

Hiroyuki Tada ◽

Hidetoshi Shimauchi ◽

Haruhiko Takada ◽

Kenji Matsushita

Keyword(s):

Epithelial Cells ◽

Porphyromonas Gingivalis ◽

Periodontal Diseases ◽

Gingival Epithelial Cells ◽

Human Gingival Epithelial Cells

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Porphyromonas gingivalis Impairs Oral Epithelial Barrier through Targeting GRHL2

Journal of Dental Research ◽

10.1177/0022034519865184 ◽

2019 ◽

Vol 98 (10) ◽

pp. 1150-1158 ◽

Cited By ~ 5

Author(s):

W. Chen ◽

A. Alshaikh ◽

S. Kim ◽

J. Kim ◽

C. Chun ◽

...

Keyword(s):

Epithelial Cells ◽

Oral Mucosa ◽

Porphyromonas Gingivalis ◽

Oral Bacteria ◽

Conditional Knockout ◽

Epithelial Barrier ◽

Wild Type ◽

Oral Epithelial ◽

Junction Proteins

Oral mucosa provides the first line of defense against a diverse array of environmental and microbial irritants by forming the barrier of epithelial cells interconnected by multiprotein tight junctions (TJ), adherens junctions, desmosomes, and gap junction complexes. Grainyhead-like 2 (GRHL2), an epithelial-specific transcription factor, may play a role in the formation of the mucosal epithelial barrier, as it regulates the expression of the junction proteins. The current study investigated the role of GRHL2 in the Porphyromonas gingivalis ( Pg)–induced impairment of epithelial barrier functions. Exposure of human oral keratinocytes (HOK-16B and OKF6 cells) to Pg or Pg-derived lipopolysaccharides ( Pg LPSs) led to rapid loss of endogenous GRHL2 and the junction proteins (e.g., zonula occludens, E-cadherin, claudins, and occludin). GRHL2 directly regulated the expression levels of the junction proteins and the epithelial permeability for small molecules (e.g., dextrans and Pg bacteria). To explore the functional role of GRHL2 in oral mucosal barrier, we used a Grhl2 conditional knockout (KO) mouse model, which allows for epithelial tissue-specific Grhl2 KO in an inducible manner. Grhl2 KO impaired the expression of the junction proteins at the junctional epithelium and increased the alveolar bone loss in the ligature-induced periodontitis model. Fluorescence in situ hybridization revealed increased epithelial penetration of oral bacteria in Grhl2 KO mice compared with the wild-type mice. Also, blood loadings of oral bacteria (e.g., Bacteroides, Bacillus, Firmicutes, β- proteobacteria, and Spirochetes) were significantly elevated in Grhl2 KO mice compared to the wild-type littermates. These data indicate that Pg bacteria may enhance paracellular penetration through oral mucosa in part by targeting the expression of GRHL2 in the oral epithelial cells, which then impairs the epithelial barrier by inhibition of junction protein expression, resulting in increased alveolar tissue destruction and systemic bacteremia.

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