Identification of interspecies interactions affecting Porphyromonas gingivalis virulence phenotypes

Abstract

Background: Periodontitis is recognized as a complex polymicrobial disease, however, the impact of the bacterial interactions among the 700-1,000 different species of the oral microbiota remains poorly understood. We conducted an in vitro screen for oral bacteria that mitigate selected virulence phenotypes of the important periodontal pathogen, Porphyromonas gingivalis.

Method: We isolated and identified oral anaerobic bacteria from subgingival plaque of dental patients. When cocultured with P. gingivalis W83, specific isolates reduced the cytopathogenic effects of P. gingivalis on oral epithelial cells.

Result: In an initial screen of 103 subgingival isolates, we identified 19 distinct strains from nine species of bacteria (including Actinomyces naeslundii, Streptococcus oralis, Streptococcus mitis, and Veilonella dispar) that protect oral epithelial cells from P. gingivalis-induced cytotoxicity. We found that some of these strains inhibited P. gingivalis growth in plate assays through the production of organic acids, whereas some decreased the gingipain activity of P. gingivalis in coculture or mixing experiments.

Conclusion: In summary, we identified 19 strains isolated from human subgingival plaque that interacted with P. gingivalis, resulting in mitigation of its cytotoxicity to oral epithelial cells, inhibition of growth, and/or reduction of gingipain activity. Understanding the mechanisms of interaction between bacteria in the oral microbial community may lead to the development of new probiotic agents and new strategies for interrupting the development of periodontal disease.

Keywords: bacterial interactions; cytotoxicity; gingipain; oral pathogen; virulence.

Fig. 1

Effects of Porphyromonas gingivalis (Pg) on oral epithelial cells. (A) Pg W83, Streptococcus oralis 180B (So180B), or Pg+So180B were added to OKF6/TERT2 cells. Cocultures were allowed to incubate in 5% CO₂ at 37°C for 16 hr and then examined by blinded investigators. (B) OKF6/TERT-2 cells were grown to confluence and used in the wound-healing assay. A linear streak was made in the cell monolayer. Pg, So180B, or Pg+So180B were added to the cultures at MOI=100. After incubation for 2 hrs, the plates were washed to remove bacteria and fresh media was added. The cultures were incubated for 16 hrs and read by blinded investigators.

Fig. 2

Effect of oral bacterial isolates on Porphyromonas gingivalis (Pg) growth. Broth cultures of 19 clinical strains identified as inhibitory to Pg cytotoxicity were spotted on blood agar inoculated with a lawn of Pg and incubated anaerobically at 37°C for 48 hrs. The growth of Pg was inhibited near the colonies of 6 of 19 strains including (A) Streptococcus intermedius and Actinomyces naeslundii. (B) Growth inhibition was measured as the size of the clear zone (in mm) between the edge of the colony and the lawn of P. gingivalis. Bars represent the average of triplicate measurements confirmed in three independent experiments. Abbreviations used: An, Actinomyces naeslundii; Pa, Propionibacterium acnes; Si, Streptococcus intermedius; Smi, Streptococcus mitis; Sc, Streptococcus constellatus; So, Streptococcus oralis; Streptococcus sanguinis; Vd, Veillonella dispar. *less than 98% 16S rRNA sequence similarity to the closest type strain.