Fusobacterium nucleatum Invades Primary Normal Human Gingival Fibroblasts

Background: Fusobacterium nucleatum is an opportunistic, strictly anaerobic, Gram-negative pleomorphic rod involved in periodontal diseases and known to adhere to and infect human gingival epithelial cells in vivo and in vitro. Little is known, however, about the interactions of the bacterium with human gingival fibroblasts. Objective: The aim of this study was to examine the ability of F. nucleatum to invade primary normal human gingival fibroblasts. Methods: Monolayers of primary normal human gingival fibroblasts obtained from biopsies were co-cultured for 1h, 3h and 24h, respectively, with ATCC strains of F. nucleatum at a multiplicity of infection (cell : bacterium ratio) of 1:100. Both transmission and scanning electron microscopy together with confocal laser scanning were used to investigate internalized bacteria. For visualization with the confocal microscope, F. nucleatum and fibroblasts were labeled with different fluorescent dyes before the co-culture. Results: Scanning electron microscopy showed bacteria entering the fibroblasts already after 1h and intracellular bacteria after the other time intervals of co-culture. Transmission electron microscopy demonstrated bacteria residing within vacuoles in the cytoplasm of the primary human gingival fibroblasts co-cultured with F. nucleatum strains ATCC 10953 and ATCC 25586. No similar structures could be detected in the cytoplasm of fibroblasts grown alone. Confocal microscopy showed that most of the internalized bacteria were located at the basal region of the cytoplasmic filopodia at 3h and perinuclearly at 24h. Conclusion: F. nucleatum was capable of invading normal human gingival fibroblasts grown in monolayers. Since fibroblasts are crucial for the maintenance of the extracellular matrix of the periodontium and may play an important role in tissue destruction in periodontitis, our in vitro findings may be of clinical significance.