Impact Of Pathogenicity On Porphyromonas Gingivalis And Aggregatibacter Actinomycetemcomitans By Fusobacterium Nucleatum

Background and Objective:Periodontitis is an inflammatory disease in the oral cavity induced by multiple bacterial infections, which can cause destruction of the tooth-supporting tissues and then lead to tooth loss. According to previous opinions, bacterial infections in the periodontium are ascribed to the interactions between oral bacteria and their surrounding epithelium. Therefore, it is necessary to clarify the interactions between different periodontal bacteria and host cells.At present,300 or more bacterial species were found in subgingival plaque, and they have been divided into six distinct complexes of closely related species. Bacteria including Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans(A. actinomycetemcomitans) identified to be associated with periodontal lesions are classed into "red complex" and "green complex". However, they are usually detected in the presence of bacteria in "orange complex", such as Fusobacterium nucleatum (F. nucleatum). F. nucleatum, one of the oral commensal bacterial species, has been shown to be able to connect with many other species of oral bacteria, serving as a bridge between the former colonizers and the latter periodontopathic bacteria and thus play an essential role in the development of dental plaque via affecting the species composition of dental plaque. In addition, F. nucleatum has been reported to interact with both P. gingivalis and A. actinomycetemcomitans.Like other infectious diseases, the adhesion and subsequent invasion to epithelial surfaces by pathogens are critical steps in the initiation of periodontitis. Invasive capacity of pathogens has been suggested as a potential pathogenic factor in periodontitis for decades. Due to the interactions between bacterial species, the coinfection of P. gingivalis or A. actinomycetemcomitans with F. nucleatum may influence the adhering and invasive capacity to human gingival epithelial cells.In response to bacteria, gingival epithelial cells serve as physical barrier and also function as sensors for detecting the presence of bacteria. The contact between bacteria and epithelium stimulates the expression of various immune response mediators from epithelial cells. Among these mediators, interleukin-8 (IL-8),a potent known proinflammatory chemokine which can induce and activate neutrophils and human beta-defensin-2 (hBD-2), an antimicrobial peptide, play important roles in both innate and adaptive immunity. Since there are different effects of bacteria in the orange-complex with that in the red-complex bacteria on the innate immune responses, the interactions between these two complexes may have synergistic impact on immune regulation in gingiva including modulating the expression of IL-8 and hBD-2.This study was conducted to investigate effects of coinfection of P. gingivalis or A. actinomycetemcomitans with F. nucleatum on their adhering and invasive capacity to human gingival epithelial cells as well as the expression of interleukin-8 (IL-8) and human beta-defensin-2 (hBD-2) in human gingival epithelial cells P. gingivalis and A. actinomycetemcomitans were tested for their ability to attach and invade a human gingival epithelial cell line (Ca9-22) alone or coinfecting with F. nucleatum. Also, expression levels of IL-8 and hBD-2 were detected respectively using enzyme linked immunosorbent assay (ELISA) and real-time reverse transcription PCR (RT-PCR) when Ca9-22 cells were infected with P. gingivalis and A. actinomycetemcomitans alone or coinfecting with F. nucleatum.Methods:1.The effects of adhering and invasive abilities to Ca9-22 cells infected with P. gingivalis and A. actinomycetemcomitans alone or coinfecting with F. nucleatum.Ca9-22 cells were seeded in a 12-well flat-bottom culture plate at a cell density of 2.0×105 cells per well. Before infection, the cells were washed twice with phosphate-buffered saline (PBS, pH 7.4) and incubated for 2 h in MEM without antibiotics. The three bacterial strains were inoculated into brain heart infusion (BHI) broth (Sigma, USA) supplemented with 0.5% of yeast extract, hemin (5μg/ml) and menadione (0.5μg/ml), and grown for 2 days until the OD660nm reached 1.0. After washed with PBS, bacterial cells were resuspended in MEM. Bacterial suspensions (2.0×107 cells per well) were added to confluent Ca9-22 monolayers and incubated at 37℃ in 5% CO2 incubator.In the attachment assay, the bacteria were incubated with the monolayers for 1 h, and then the monolayers were washed with PBS four times to remove the unattached bacteria. Subsequently,1 ml of sterile distilled water was added to each well to lyse cells for 90 min. Lysates diluted 1,000 times were then plated on Brain Heart Infusion agar-Supplemented (BHI-S) blood agar plates (100μl per plate) and incubated anaerobically at 37℃ for 10 days.In the invasion assay, bacteria were incubated with the monolayers for 4 h. Following incubation, the monolayers were washed with PBS four times to remove unattached bacteria and incubated with MEM containing 200μg/ml of metronidazole and 300μg/ml of gentamicin for 1h to kill external adherent cells. After exposure to antibiotic, monolayers were washed twice with PBS, then cells were lysed in 1 ml of sterile distilled water per well for 90 min. Lysates were diluted 100 times and plated on BHI-S blood agar plates (100μl per plate), then incubated anaerobically at 37℃ for 10 days.Colony-Forming Units (CFU) of attached and invasive organisms was then enumerated following incubation. Attachment and invasion efficiency was expressed as the percentage of bacteria retrieved following cell lysis relative to the total number of bacteria initially added.2.The expression levels of IL-8 and hBD-2 in Ca9-22 cells infected with P. gingivalis and A. actinomycetemcomitans alone or coinfecting with F. nucleatum.To determine the amount of IL-8 secreted by Ca9-22 cells, the bacterial suspensions (2.0×107 cells per well) were added to confluent Ca9-22 monolayers and incubated at 37℃ in 5% CO2 for 4 h. Then the supernatant was collected and stored at -20℃. A standard enzyme linked immunosorbent assay (ELISA) was used to quantify the concentration of IL-8 in the culture supernatant. A mouse monoclonal antibody specific for IL-8 (1:100, Dako) was used as the capture antibody. The recombinant IL-8 or the IL-8 in the test samples captured by the mouse antibody was detected by the addition of polyclonal rabbit antibodies specific for IL-8, followed by the addition of biotinylated sheep anti-rabbit IgG (1:200, Dako) and then streptavidin-horseradish peroxidase (HRP) and the HRP substrate 2,2’-azinobis (3-ethylbenzthiazolinesulfonic acid). All incubations were carried out for 45 min at 37℃. The concentrations of IL-8 in the test samples were extrapolated from a standard curve generated with known concentrations of recombinant beta-hemolytic streptococcal (BHS) and IL-8.Total RNA (2μg) from Ca9-22 cells was subjected to reverse transcription with (dT)18 and Superscript Ⅱ enzyme (Invitrogen) in a 25-μl reaction mix at 42℃ for 1 h. Real time-PCR was performed in a 20-μl reaction mix containing 1μl of template cDNA, SYBR Premix Ex Taq, ROX Reference Dye (Sigma, USA), and each primer (0.2μM). Primer sequences used were:GAPDH forward, 5’-CAGCCTCAAGATCATCAGCA-3’ and reverse, 5’-CCATCCACAGTCTTCTGGGT-3’; hBD-2 forward, 5’-AGACTCAGCTCCTGGTGAAGCTC-3’ and reverse, 5’-TGGCTCCACTCTTAAGGCAGGTA-3’. All primers were designed to amplify at least two exons in order to prevent the amplification of contaminating gDNA. Amplification was performed in a fluorescence thermocycler (StepOne Plus) under the following conditions:initial denaturation at 94℃ for 1 min, followed by 40 cycles of denaturation at 95℃ for 15 s, annealing at 62℃ for 15 s, and elongation at 72℃ for 33 s. The specificity of the PCR product was verified by melting curve analysis and examination on a 3% agarose gel. The housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was amplified in parallel with the gene of interest. Relative copy numbers compared to GAPDH were calculated by using 2-ΔCT.Results:1.The effects of adhering and invasive abilities to Ca9-22 cells infected with P. gingivalis and A. actinomycetemcomitans alone or coinfecting with F. nucleatum.The attachment efficiency of P. gingivalis, A. actinomycetemcomitans and F. nucleatum was 0.97±0.38%,2.62±0.58% and 1.06±0.30%, respectively. Coinfection of P. gingivalis with F. nucleatum significantly boosted attachment efficiency of P. gingivalis to Ca9-22 cells by 2-fold (P<0.05). The attachment efficiency of A. actinomycetemcomitans to Ca9-22 cells in the presence of F. nucleatum was approximately increased by 178.24% over A2-treatment (P<0.05). These results indicated that the enhanced attachment of P. gingivalis and A. actinomycetemcomitans after coincubation with F. nucleatum can be inhibited by the inhibitor of F. nucleatum adhesion/invasion galactose.The invasion efficiency of P. gingivalis, A. actinomycetemcomitans and F. nucleatum was 0.35±0.08%,1.01±0.15% and 0.44±0.11%, respectively. Invasion efficiency of P. gingivalis and A. actinomycetemcomitans to Ca9-22 cells significantly increased by 251.43% and 207.92%, respectively, in the presence of F. nucleatum than without F. nucleatum (P<0.05). These results indicated that F. nucleatum could enhance the invasion ability of P. gingivalis and A. actinomycetemcomitans to Ca9-22 cells while this effect could be inhibited by galactose.2.The expression levels of IL-8 and hBD-2 in Ca9-22 cells infected with P. gingivalis and A. actinomycetemcomitans alone or coinfecting with F. nucleatum.In mono-microbial infection experiments, IL-8 production in Ca9-22 cells was highest (428.75 pg mL-1) in A3-treatment and conversely lowest (386.66 pg mL-1) in Al-treatment. In poly-infection experiments, IL-8 production in B1- and B2-treatments was respectively reduced by 6.12% and 8.05% than that in A3-treatment. In C1-C4 treatments, IL-8 production was slightly higher than that in B1-B4 treatments.In mono-microbial infection experiments, the expression level of hBD-2 mRNA in Ca9-22 cells was highest (0.48) in A3-treatment and lowest (0.26) in A1-treatment. In poly-infection experiments, expression level of hBD-2 mRNA in B1-and B2-treatments was respectively decreased by 66.94% and 51.39% than that in A3-treatment. Meanwhile, in C1-C4 treatments, hBD-2 mRNA expression was all accordingly higher than that in B1-B4 treatments.Conclusion:1、Coinfection with F. nucleatum can enhance adhesion and invasion of P. gingivalis and A. actinomycetemcomitans to Ca9-22 cells.2、P. gingivalis and A. actinomycetemcomitans coinfected by F. nucleatum downregulate the expression of IL-8 and hBD-2.