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. 2024 Dec 2;22:609–616. doi: 10.3290/j.ohpd.b5866430

Commensal Neisseria Inhibit Porphyromonas Gingivalis Invasion of Gingival Epithelial Cells

Shota Fukuda a, Tomoki Akatsu b, Akihiko Fujii c, Sawako Kawano d, Yoshihiko Minegishi e, Noriyasu Ota f
PMCID: PMC12099076  PMID: 39620245

Abstract

Purpose

Periodontal disease is caused by periodontal invasion by pathogens such as Porphyromonas gingivalis. Although recent metagenomic analyses have shown that oral commensal bacteria are abundant in the mouth of healthy individuals, few studies have experimentally verified the benefits and functions of oral commensal bacteria in periodontal diseases. In this study, we focused on Neisseria among the oral commensal bacteria and aimed to experimentally verify its effects on P. gingivalis invasion.

Materials and Methods

We evaluated the inhibitory effect of Neisseria spp. on P. gingivalis invasion using a flow cytometry-based invasion assay and analysed bacterial interactions by visualisation using scanning electron microscopy. Furthermore, we constructed a new experimental pre-mixed culture system that reproduced the interaction environment to evaluate the relevance of this interaction in invasion inhibition.

Results

Flow cytometry-based invasion assays showed that all Neisseria spp. inhibited P. gingivalis invasion, with Neisseria mucosa and Neisseria elongata being particularly effective. Interaction analysis using scanning electron microscopy showed that N. mucosa and N. elongata, which have strong inhibitory effects on P. gingivalis invasion, interacted with P. gingivalis at high frequencies.

Conclusion

Commensal Neisseria was found to exert a beneficial function by directly interacting with P. gingivalis and inhibiting its invasion of gingival epithelial cells. These results suggest that Neisseria, as a probiotic or synbiotic oral commensal, may represent an innovative approach to preventing periodontal disease.

Keywords: Neisseria, oral probiotics, invasion inhibition, periodontitis

Periodontal disease is caused by bacterial infection, 7 which leads to periodontal tissue destruction through alveolar bone resorption and inflammation, resulting in tooth loss. 5,31 Porphyromonas gingivalis — a gram-negative, anaerobic, black-pigmented bacterium — is known to be a representative periodontal pathogen and a component of the ‘red complex’ with Treponema denticola and Tannerella forsythia. 18 P. gingivalis has been reported to infect gingival epithelial cells, 52 resulting in epithelial cell dysfunction and periodontal tissue destruction. Also, virulent factors of P. gingivalis, such as the capsule, outer membrane proteins, lipopolysaccharides, proteases such as gingipains, collagenases, haemolysin, trypsin proteases, hemagglutinins, and fimbriae, are involved in colonisation and invasion. 14,25,34,53,54 During an infection, P. gingivalis can penetrate deep into epithelial cells, eventually invading tissues 1 and circulating throughout the body via the red blood cells. 6 Systemically circulating P. gingivalis has been reported to invade the brain and contribute to the progression of Alzheimer’s disease, 36 invade human colonic artery endothelial cells, contribute to atherosclerosis, 37 infect immortalised human oral epithelial cells, and induce oral squamous cell carcinoma. 15 Therefore, P. gingivalis infection is not only a serious factor in the progression of periodontal disease but can also cause systemic diseases.

Over 700 bacterial species inhabit the oral cavity, including both periodontal pathogens, such as P. gingivalis, and non-pathogenic commensal bacteria. 2,44 Recent metagenomic analyses have shown that Neisseria, a gram-negative bacteria, is more abundant in healthy individuals than in individuals with periodontal disease. 8,21,49,50 More than 20 Neisseria spp. exist 29 including Neisseria mucosa, Neisseria sicca, and Neisseria elongata in plaques, and Neisseria flavescens, Neisseria flava, and Neisseria subflava in saliva. 13 These reports indicate that oral commensal Neisseria may be beneficial in maintaining oral health. However, no studies have experimentally verified the effect of Neisseria on periodontal disease and periodontal pathogens, and it is unclear whether Neisseria plays a beneficial role against periodontal disease. Therefore, we investigated the effects of Neisseria spp. on P. gingivalis invasion using in-vitro evaluation systems and microscopic visualisation techniques.

MATERIALS AND METHODS

Cells and Culture Conditions

The human gingival epithelial cell line, Ca9-22 (JCRB0625), is an established transformed human gingival cell line that has been used in previous studies 43 as a culture model of oral epithelial cells; the cell line was obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan). Ca9-22 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (GibcoTM Life Technologies, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS) (Sigma-Aldrich, St Louis, MO, USA) and 1% penicillin-streptomycin (GibcoTM Life Technologies, Carlsbad, CA, USA) at 37°C in 5% CO2. The cells were used in this study based on information from previous studies 43 that tested similar in-vitro evaluation systems.

Bacterial Strains and Growth Conditions

The periodontal pathogen strains used in this study were P. gingivalis strain ATCC 33277 and Fusobacterium nucleatum strain ATCC 23726. These bacteria were cultured on a blood agar plate (BD BBLTM anaerobic Columbia RS blood agar plate, BD Biosciences, Franklin Lakes, NJ, USA) for colony formation, and then 5–10 colonies were transferred to Gifu Anaerobic Medium (GAM) broth (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5.0 µg mL–1 Hemin, 17.4 µg mL–1 K2HPO4, and 1.0 µg mL–1 vitamin K at 37°C under anaerobic conditions. The Neisseria strains used in this study were N. mucosa strain JCM 12992, N. elongata strain ATCC 25295, N. sicca strain ATCC 29256, N. flava strain ATCC 14221, N. flavescens strain ATCC 13120, and N. subflava strain ATCC 49275. These bacteria were cultured on Brain Heart Infusion (BHI) agar (BD Biosciences, Franklin Lakes, NJ, USA), and then 5–10 colonies were transferred to BHI broth (BD Biosciences, Franklin Lakes, NJ, USA) at 37°C in 5% CO2.

P. gingivalis and F. nucleatum were pre-incubated for 24 h and then inoculated into fresh GAM broth supplemented with 5.0 µg mL–1 Hemin, 17.4 µg mL–1 K2HPO4, and 1.0 µg mL–1 vitamin K for a further 24 h with the addition of 1/1,000 volume of the pre-incubated culture at 37°C under anaerobic conditions. Neisseria spp. were pre-incubated for 48 h and then inoculated into fresh BHI broth for a further 48 h with the addition of 1/100 volume of the pre-incubated culture at 37°C in 5% CO2.

Carboxylfluorescein Diacetate Succinimidyl Ester Labelling of P. gingivalis

P. gingivalis cultures were centrifuged at 4,500 g for 10 min at 24°C, and the bacteria were collected. The recovered bacteria were washed with phosphate-buffered saline (PBS) (DPBS without calcium and magnesium; GibcoTM Life Technologies, Carlsbad, CA, USA) and serum-free DMEM at 4,500 g for 5 min at 24°C. The bacteria were adjusted to OD600 = 2.0 and incubated in 10 µM carboxylfluorescein diacetate succinimidyl ester (CFSE) (Wako, Osaka, Japan) dissolved in serum-free DMEM at 37°C in the dark for 30 min under anaerobic conditions. CFSE-labelled P. gingivalis cells were prepared and washed twice with serum-free DMEM.

Epithelial Cell Invasion Assay

Ca9-22 cells were seeded in 12-well plates at 0.2×10 6 cells per well and incubated for 48 h. The medium was removed and substituted with the serum-free DMEM after washing with PBS. The multiplicity of infection (MOI) was calculated based on the number of cells per well when they reached confluence. Then, Neisseria spp. (MOI = 100, 200, 500) or F. nucleatum (MOI = 100, 200) suspended in serum-free DMEM were added and pre-incubated for 1 h at 37°C in 5% CO2. CFSE-labelled P. gingivalis (MOI = 500) was added and incubated for 2 h at 37°C in 5% CO2 under 80 rpm shaking conditions (n = 3).

Flow Cytometry Analysis

After infecting Ca9-22 cells with CFSE-labelled P. gingivalis, unadhered bacteria were removed by washing twice with PBS. External adherent bacteria were then killed by incubation in DMEM containing 300 µg mL–1 of gentamicin (Wako, Osaka, Japan) and 200 µg mL–1 of metronidazole (Wako, Osaka, Japan) for 1 h. This concentration of antibiotics was sufficient to kill 10 8 bacteria mL–1 in 1 h at 37°C. After antibiotic exposure, the cells were detached using 0.25% Trypsin-EDTA (GibcoTM Life Technologies, Carlsbad, CA, USA) after washing twice with PBS. Then, DMEM (containing 1% FBS) was added, and the cells were collected in 1.5 mL tubes. The collected cells were centrifuged at 400 g for 5 min at 4°C. The supernatant was then removed. Cells were pipetted with 4% paraformaldehyde phosphate buffer (Wako, Osaka, Japan) and fixed for 15 min at 4°C in the dark. After fixation, PBS (containing 2% FBS) was added, the cells were mixed and centrifuged at 600 g for 5 min at 4°C, and the cells were resuspended in PBS (containing 2% FBS) for analysis. Flow cytometry (BD FACSVerseTM, BD Biosciences, Franklin Lakes, NJ, USA) was used for analysis.

Antibacterial Assay

The bacteria were prepared in the same manner as that for the epithelial cell invasion assay without CFSE labelling. The same amounts of P. gingivalis and Neisseria spp. used in the epithelial cell invasion assay were added to 12-well plates without Ca9-22 cells and incubated for 2 h. After incubation, the culture medium was collected in 1.5 mL tubes and mixed well. The collected culture was diluted, plated on a blood agar plate, and incubated at 37°C under anaerobic conditions for 2 days (under such anaerobic conditions, Neisseria spp. do not colonise during this experiment). We determined the number of viable P. gingivalis by counting the colonies (n = 3).

Scanning Electron Microscopy

Semi-confluent Ca9-22 cells on 15-mm-diameter glass coverslips were co-cultured with P. gingivalis (MOI = 500) and/or Neisseria spp. (MOI = 500) without antibiotics for 30 min. After infecting Ca9-22 cells with P. gingivalis, unadhered bacteria were removed by washing with 10 mM HEPES buffer (pH 7.4) (GibcoTM Life Technologies, Carlsbad, CA, USA) and fixed in 2.5% glutaraldehyde (Wako, Osaka, Japan) in 10 mM HEPES buffer for 2 h at 4°C. Following serial dehydration, all samples were coated with gold using a smart coater (DII-29010SCTR; Japan Electron Optics Laboratory, Tokyo, Japan). Ca9-22 cells and bacteria were visualised using scanning electron microscopy (SEM) (JSM-6510; Japan Electron Optics Laboratory, Tokyo, Japan) at an accelerating voltage of 10.0 kV to directly observe bacterial–bacteria and bacterial–host-cells interactions.

Pre-mixed Culture and Simultaneous Culture Systems

For the pre-mixed culture system, the prepared Neisseria spp. (MOI = 500) or F. nucleatum (MOI = 200) was mixed with CFSE-labelled P. gingivalis (MOI = 500) for 1 h at 37°C under 80 rpm shaking conditions. Then, the mixture was added to Ca9-22 cells and incubated for 2 h at 37°C.

For the simultaneous culture system, the bacteria were prepared according to the procedure used for the epithelial cell invasion assay. Neisseria spp. (MOI = 500) or F. nucleatum (MOI = 200) and CFSE-labelled P. gingivalis (MOI = 500) were added to Ca9-22 cells simultaneously and incubated for 2 h at 37°C without pre-incubation. The same procedure used for the epithelial invasion assay was used for subsequent sample preparation and flow cytometric analysis (Neisseria spp.; n = 6, F. nucleatum; n = 3).

Statistical Analysis

The mean fluorescence intensity was calculated from the fluorescence intensity obtained from fluorescein isothiocyanate channels of 500,000 cells gated according to forward scatter and side scatter, and the fluorescence intensity was used to assess P. gingivalis invasion. To compare the invasive ability of different strains, the invasion index was calculated as follows: [mean fluorescence intensity (MFI) of infected cells–MFI of negative control cells]/MFI of cells infected with CFSE-labelled P. gingivalis. Data from the epithelial cell invasion and antibacterial assays were analysed using Dunnett’s test to determine whether there were differences with and without Neisseria spp. The data from the pre-mixed culture system were analysed using Tukey–Kramer’s test to determine whether there were differences between the simultaneous and pre-mixed culture systems. These data are shown as relative mean ± standard deviation with P. gingivalis alone as 1.0, and antibacterial assay results are shown as raw colony forming unit values ± standard deviation. A P-value < 0.05 was considered statistically significant.

RESULTS

In-vitro Evaluation of the Effect of Neisseria spp. on P. gingivalis Invasion

To evaluate the effect of Neisseria spp. on P. gingivalis gingival epithelial cell invasion, we established an in-vitro evaluation system based on previous studies. 43 When CFSE-labelled P. gingivalis was co-cultured with Neisseria spp. (MOI = 200, 500), all Neisseria spp. decreased the fluorescence intensity (Figs 1a–1f), especially for N. mucosa and N. elongata (Figs 1a and 1b). Conversely, when CFSE-labelled P. gingivalis was co-cultured with F. nucleatum (MOI = 200), the fluorescence intensity was increased (Fig 1g). The co-culture of CFSE-labelled P. gingivalis and F. nucleatum (MOI = 500) showed high cytotoxicity at 2 h of infection and did not reach analysable cell numbers (data not shown). Also, we performed an antibacterial assay under anaerobic conditions in the absence of cells, exploiting the inability of Neisseria spp. to grow, and evaluated whether Neisseria spp. has antibacterial activity against P. gingivalis (Fig 1h). No difference was observed in the number of viable P. gingivalis colonies between P. gingivalis alone and P. gingivalis mixed with Neisseria spp. (Fig 1h).

Neisseria spp. and P. gingivalis Interaction

To investigate the mechanism of fluorescence intensity suppression by Neisseria spp., we used SEM to visualise the interactions between Neisseria spp. and P. gingivalis on the cell surface. In the analyses, N. mucosa and N. elongata — which effectively inhibited P. gingivalis invasion in the invasion assay — were compared with N. sicca, which is known to be present in plaques as well as N. mucosa and N. elongata. 13 For each bacterium, P. gingivalis was observed as a coccus or bacillus (Fig 2a), N. mucosa and N. sicca as a diplococcus (Figs 2b and 2d), and N. elongata as a bacillus (Fig 2c). N. mucosa and N. elongata, which showed high inhibition of invasion, interacted with P. gingivalis at most sites (Figs 2e and 2f). In contrast, N. sicca, which inhibited P. gingivalis invasion less effectively than N. mucosa and N. elongata did, was located around P. gingivalis, but did not interact with P. gingivalis in most regions (Fig 2g).

Effect of Interaction in P. gingivalis Invasion Ability

To clarify the relationship between fluorescence intensity suppression and bacterial interactions, a pre-mixed culture system was constructed to reproduce the interaction environment (Fig 3a). For comparison, we also tested how the effect changes in the interaction environment using the simultaneous culture system, which was almost the same as that in the invasion assay (Fig 3b). In the pre-mixed culture system, N. mucosa, N. elongata, and N. sicca decreased the fluorescence intensity by 34%, 57%, and 43%, respectively (Figs 3c–3e). Conversely, F. nucleatum increased the fluorescence intensity by 26% (Fig 3f). In the simultaneous culture system, N. mucosa, N. elongata, and N. sicca decreased the fluorescence intensity by 35%, 38%, and 13%, respectively (Figs 3c–3e). In contrast, F. nucleatum increased the fluorescence intensity by 6% (Fig 3f).

DISCUSSION

In this study, we aimed to experimentally verify commensal Neisseria effects on P. gingivalis invasion. The results show that all Neisseria spp. inhibited P. gingivalis invasion of gingival epithelial cells under conditions where F. nucleatum promoted P. gingivalis infection as in previous studies (although with a slight increase in this study), and bacterial interaction may be involved. However, the exact mechanism of this interaction with P. gingivalis to inhibit invasion remains unclear. The components involved in P. gingivalis virulence, such as fimbriae and gingipains are important for infection 14,25,34,53,54 ; particularly, fimbriae-related virulence has also been extensively examined using strains with defective fimbriae. 17 Therefore, further detailed studies, such as utilising P. gingivalis mutants lacking fimbriae, are needed to gain a more comprehensive understanding of the interaction. Also, recent studies have shown that P. gingivalis and F. nucleatum affect periodontitis by inducing epigenetic changes in gingival epithelial cells. 26 Therefore, it is also necessary to assess the effect of Neisseria spp. on these epigenetic changes. In addition, the effects of P. gingivalis on Neisseria spp. must be considered. Therefore, when assessing bacterial interactions, it is necessary to consider both the effects of virulence factors and epigenetic changes.

Importantly, in the pre-mixed culture system, a further inhibitory effect on invasion was not observed for N. mucosa. This suggests that N. mucosa inhibits P. gingivalis invasion via a different mechanism than that of N. elongata and N. sicca. Neisseria have been shown to have the ability to reduce NO₃– and/or NO₂–. 20 Notably, N. mucosa is the only Neisseria species that has been reported to possess the ability to reduce not only NO₂– to NO but also NO₃– to NO₂–, unlike other Neisseria spp., including N. elongata and N. sicca. 4 Whether this ability to reduce NO₃– and/or NO₂– plays a role in the inhibition of invasion is unknown; however, N. mucosa may have properties distinct from other Neisseria spp. that increase its effectiveness in inhibiting invasion. Therefore, further assessment of the other properties of N. mucosa and the mechanism by which it inhibits P. gingivalis invasion is required.

In this study, we experimentally demonstrated the beneficial role of Neisseria spp. for periodontal disease for the first time. Several studies have reported that Neisseria may have beneficial functions for diseases other than periodontal disease. N. elongata is more abundant at the supragingival margins, 38 and N. subflava is more abundant on the tongue 55 in healthy individuals than in individuals with caries. There have also been many reports of low Neisseria levels in patients with oral cancer, 10,16,51,56 and species-level analyses have shown that N. sicca and N. flavescens are particularly low 3,30,35 in those cases. Additionally, Neisseria spp. has been shown to decrease the proliferation rate of carcinoma cells, 19 and N. sicca plays a role in maintaining genomic stability in the control of oral cancer. 45 Low levels of Neisseria, especially N. elongata, 9 have also been reported in patients infected with influenza A pdm09 virus 27 and severe acute respiratory syndrome coronavirus 2. 11,12,32 It has also been confirmed that Neisseria is low in patients with oesophageal cancer 28 and that N. mucosa is halved in the oral cavity of patients with inflammatory bowel disease. 42 These reports suggest that commensal Neisseria may be an important oral probiotic against periodontal disease, dental caries, oral cancer, and systemic diseases.

Recent studies have shown that probiotics or synbiotics as new oral care approaches have been proposed as a promising preventative strategy.39–41,46 There have been some reports, on the effects of Lactobacillus and Bifidobacterium on P. gingivalis. For example, Limosilactobacillus fermentum ALAL020 produces cyclic peptides and shows antibacterial activity, 24 Lacticaseibacillus rhamnosus L8020 inhibits the accumulation of periodontal disease-related pathogens, 33 and Bifidobacterium dentium and Bifidobacterium longum specifically reduce the number of viable P. gingivalis. 23 However, there have been few reports on the use of oral commensal bacteria as probiotics. This study evaluated, for the first time, the effects of Neisseria spp. on P. gingivalis invasion using an in-vitro evaluation system and SEM. In addition, since commensal Neisseria are significantly increased in the oral cavity by the ingestion of nitrate, 39,47,48 it is conceivable that this could be a possible approach to prevent periodontal disease through the effects of synbiotics that combine nitrate and beneficial Neisseria. In other words, we demonstrated the possibility of using oral Neisseria spp. — a commensal bacterium — for the prevention of periodontal disease, unlike conventional probiotics such as Lactobacillus and Bifidobacterium.

Our study has some limitations. First, the absolute number of P. gingivalis that invaded into Ca9-22 cells cannot be estimated based on the fluorescence intensity of our invasion assay. Methods to distinguish adherent bacteria from invading bacteria using antibodies instead of fluorescent labelling have been reported, 22 and such evaluation should be considered in future research. Second, this study evaluated the effect of Neisseria on P. gingivalis in-vitro; however, the differences between the present experimental system and real-life clinical conditions are not yet clear. The oral cavity is a complex environment, hosting several bacterial species. Therefore, to verify the effect of Neisseria spp. in the oral cavity more reliably and in detail, in-vivo and human studies are also necessary. Third, we have only evaluated the inhibitory effect of P. gingivalis invasion by Neisseria spp. on one cell line (Ca9-22 cells) and one P. gingivalis strain (type strain: ATCC 33277). Also, the effects of Neisseria on P. gingivalis were assessed; however, the effects of P. gingivalis on Neisseria were not assessed. Therefore, it is necessary to understand these interactions in more detail by examining the interactions in multiple cell lines and P. gingivalis strains and assessing the effects of P. gingivalis on Neisseria.

In this study, we found that commensal Neisseria inhibited P. gingivalis invasion of gingival epithelial cells. To our knowledge, this is the first study to report the effects of Neisseria on periodontal pathogens. Furthermore, we found that N. elongata and N. sicca inhibit P. gingivalis invasion through direct interactions. However, the mechanisms by which N. mucosa inhibits P. gingivalis invasion remain to be elucidated and require further analysis. This study suggests that Neisseria, as a probiotic or synbiotic commensal bacteria in the oral cavity, may represent a new approach for preventing periodontal disease.

Acknowledgements

This research was financially supported by the Kao Corporation. The authors would like to thank Taichi Konno (Processing Development Research, Kao Corporation) for technical assistance with the SEM experiments and Editage (http://www.editage.com) for editing and reviewing this manuscript for enhanced English language.

Fig 1.

Fig 1

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Neisseria spp. inhibition of Porphyromonas gingivalis invasion. (a) Mono- or co-infection of P. gingivalis with N. mucosa. (b) Mono- or co-infection of P. gingivalis with N. elongata. (d) Mono- or co-infection of P. gingivalis with N. sicca. (d) Mono- or co-infection of P. gingivalis with N. flava. (e) Mono- or co-infection of P. gingivalis with N. flavescens. (f) Mono- or co-infection of P. gingivalis with N. subflava. (g) Mono- or co-infection of P. gingivalis with F. nucleatum. Relative mean fluorescence intensity [rMFI] is shown as relative mean ± standard deviation with P. gingivalis alone as 1.0 (n = 3, n.s = not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, Dunnett’s test). (h) Quantification of the number of viable P. gingivalis detected in the antibacterial assay. Colony forming unit (CFU) is shown as relative mean ± standard deviation with P. gingivalis alone as 1.0 (n = 3, n.s = not significant (P > 0.05), Dunnett’s test). Nm = N. mucosa, Ne = N. elongata, Ns = N. sicca, Nf = N. flava, Nfs = N. flavescens, Nsf = N. subflava.

Fig 2.

Fig 2

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N. mucosa and N. elongata interacting with Porphyromonas gingivalis at a high frequency. (a) Scanning electron microscopy (SEM) image of P. gingivalis (scale bar = 1 µm). (b) SEM image of N. mucosa (scale bar = 0.5 µm). (c) SEM image of N. elongata (scale bar = 1 µm). (d) SEM image of N. sicca (scale bar = 0.5 µm). (e) SEM images of P. gingivalis and N. mucosa co-cultures (left image: scale bar = 5 µm, right image: scale bar = 1 µm). (f) SEM images of P. gingivalis and N. elongata (left image: scale bar = 5 µm, right image: scale bar = 1 µm). (g) SEM images of P. gingivalis and N. sicca co-culture (left image: scale bar = 5 µm, right image: scale bar = 1 µm).

Fig 3.

Fig 3

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Further inhibitory effect on invasion by Neisseria spp. in the pre-mixed culture system. (a, b) Summary of the pre-mixed culture system (PmC) and simultaneous culture system (SC) used for comparison. (c) Mono- or co-infection of P. gingivalis with N. mucosa. (d) Mono- or co-infection of P. gingivalis with N. elongata. (e) Mono- or co-infection of P. gingivalis with N. sicca. (f) Mono- or co-infection of P. gingivalis with F. nucleatum. Relative mean fluorescence intensity [rMFI] is shown as relative mean ± standard deviation with P. gingivalis alone as 1.0 (n = 3–6, n.s = not significant (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, Tukey–Kramer’s test).

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