Invasion of oral epithelial cells by<i>Actinobacillus actinomycetemcomitans</i>

Meyer, Diane H.; Lippmann, Joan E.; Fives-Taylor, Paula

doi:10.1017/cbo9780511546273.011

Cited by 71 publications

(106 citation statements)

References 66 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Yet, the complex relationship between the subgingival microbiota and the epithelium of the gingival crevice in such infections has only been partially determined. A great deal of evidence has accumulated which corroborates the ability of putative periodontal pathogens, such as Prevotella intermedia (Dorn et al, 1998), Actinobacillus actinomycetemcomitans (FivesTaylor et al, 1995;Meyer et al, 1996;Rudney et al, 2001), Porphyromonas gingivalis (Sandros et al, 1994;Lamont et al, 1995;Madianos et al, 1996), Fusobacterium nucleatum (Han et al, 2000), and several species of oral spirochaetes (Peters et al, 1999), to adhere to and invade oral epithelial tissues and cells. However, most of these studies were performed in vitro, using few reference bacterial strains, and oral epithelial cell lines, primary epithelial cell cultures or cells from the buccal mucosa.…”

Section: Introductionmentioning

confidence: 96%

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

Colombo¹,

Silva²,

Haffajee

et al. 2006

Journal of Medical Microbiology

View full text Add to dashboard Cite

Bacterial invasion of host epithelial cells plays an important role in the pathogenesis of periodontal diseases; however, the interactions between subgingival species and the gingival crevice cells are not fully understood. This study determined the prevalence of a group of oral bacterial species on or in epithelial cells derived from periodontal pockets and the gingival crevice of subjects with periodontitis. Samples of epithelial cells were obtained from 120 sites with periodontal pockets ¢4 mm and 92 periodontally healthy sites from 49 patients (mean age 46?3±1?4 years; 43 % males) with chronic periodontitis. Bacteria in or on epithelial cells were separated from unattached bacteria by Percoll density-gradient centrifugation. The presence and levels of 33 oral species were determined in epithelial cell samples by whole genomic DNA probes and the checkerboard method. The most frequently detected species were Porphyromonas gingivalis (42 %), Treponema denticola (38 %), Prevotella intermedia (37 %), Streptococcus intermedius (36 %), Campylobacter rectus (35 %), Streptococcus sanguinis (35 %) and Streptococcus oralis (34 %). Species of Actinomyces were found in low prevalence and levels. The data indicated that there were more micro-organisms on or in epithelial cells obtained from periodontal pockets than from healthy sulci; however, no significant differences regarding the percentage and level of any specific species were found between these sites. Veillonella parvula, S. oralis, Streptococcus gordonii and Streptococcus mitis tended to be more prevalent in sites without disease. These findings demonstrated that a wide range of oral species may be detected on or in crevicular epithelial cells from sites with periodontitis and from periodontally healthy sulci.

show abstract

Section: Introductionmentioning

confidence: 96%

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

Colombo¹,

Silva²,

Haffajee

et al. 2006

Journal of Medical Microbiology

View full text Add to dashboard Cite

show abstract

“…actinomycetemcomitans is a facultative, Gram-negative bacterium that has long been associated with localized aggressive periodontitis. A. actinomycetemcomitans is one of the few periodontal pathogens capable of active tissue invasion (Blix et al, 1992;Meyer et al, 1996). The bacterium expresses a number of gene products that can be defined as virulence factors based on their in vitro biological activities or similarities to products produced by other pathogens (Henderson et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells

Kang

Korostoff

Volgina

et al. 2005

Journal of Medical Microbiology

View full text Add to dashboard Cite

The periodontal pathogen Actinobacillus actinomycetemcomitans expresses a cytolethal distending toxin (CDT) that typically arrests the growth of eukaryotic cells at either the G 0 /G 1 or G 2 /M phase of the cell cycle. It was previously found that CDT failed to arrest the growth of human periodontal ligament fibroblasts (HPLFs) when grown in pure culture. In contrast, proliferation of an oral epithelial cell line was rapidly inhibited by the toxin. In this study, the feasibility of using mixed-cell cultures and cell-specific markers to evaluate the response of oral cells, when in heterogeneous populations, to CDT was established. Proliferation of epithelial cells was rapidly inhibited and the cells were selectively eliminated in co-culture with HPLFs or cementoblasts by 24-48 h postintoxication. Epithelial cells and HPLFs were detected and counted in co-cultures following cellspecific immunolabelling with antibodies against simian virus 40 large T antigen and the Ab-1 surface antigen, respectively. These results demonstrated that the activities of potential virulence factors, such as CDT, from periodontal pathogens can be successfully examined in mixed-cell cultures. This approach is especially relevant to infectious diseases that affect tissues with a diverse cellular composition, such as the periodontium.

show abstract

“…The assay is also attractive because it requires no specialist equipment, it can be tailored to work with various bacteria and cells, and can be used to study the role of both bacterial and host cell processes in invasion 9 . Indeed, since the earliest experiments, the gentamicin protection assay has been widely employed to measure host cell invasion by a range of different bacteria or even combinations of bacteria 10,11 . Whilst the core principles are the same, many subtle variations in the method have been reported.…”

Section: Discussionmentioning

confidence: 99%

Invasion of Human Cells by a Bacterial Pathogen

Edwards

Massey

2011

JoVE

View full text Add to dashboard Cite

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO 2 at 37°C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 μl tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy. Video LinkThe video component of this article can be found at https://www.jove.com/video/2693/ ProtocolThe following protocol will describe the study of endothelial cell invasion by S. aureus but can theoretically be used to study cellular invasion by any culturable bacterium. Stages specific to S. aureus and endothelial cells are indicated. Preparation of Bacteria1. Culture S. aureus strains for 4-16 h (depending on growth phase required) in 10 ml Brain-Heart Infusion (BHI) broth at 37°C in air with shaking at 200 rpm. These growth conditions are specific for S. aureus and may need to be adapted for other bacteria. 2. Wash bacteria three times in Dulbecco's Modified Eagle's medium (DMEM; Invitrogen) by alternate rounds of centrifugation at room temperature (5,000 x g , 10 min), removal of the culture supernatant and resuspension of the bacterial pellet in an equivalent volume of DMEM. Measure the optical density of the resulting suspension of bacteria which can then be adjusted as required. For S. aureus, we prepare a suspension at OD 600 = 1, which corresponds to ~10 9...

show abstract

Invasion of oral epithelial cells byActinobacillus actinomycetemcomitans

Cited by 71 publications

References 66 publications

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

Identification of oral bacteria associated with crevicular epithelial cells from chronic periodontitis lesions

Differential effect of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans on co-cultures of human oral cells

Invasion of Human Cells by a Bacterial Pathogen

Contact Info

Product

Resources

About