Tannerella forsythia scavenges Fusobacterium nucleatum secreted NOD2 stimulatory molecules to dampen oral epithelial cell inflammatory response

Abstract

The oral organism Tannerella forsythia is auxotrophic for peptidoglycan amino sugar N-acetylmuramic acid (MurNAc). It survives in the oral cavity by scavenging MurNAc- and MurNAc-linked peptidoglycan fragments (muropeptides) secreted by co-habiting bacteria such as Fusobacterium nucleatum with which it forms synergistic biofilms. Muropeptides, MurNAc-l-Ala-d-isoGln (MDP, muramyl dipeptide) and d-γ-glutamyl-meso-DAP (iE-DAP dipeptide), are strong immunostimulatory molecules that activate nucleotide oligomerization domain (NOD)-like innate immune receptors and induce the expression of inflammatory cytokines and antimicrobial peptides. In this study, we utilized an in vitro T. forsythia-F. nucleatum co-culture model to determine if T. forsythia can selectively scavenge NOD ligands from the environment and impact NOD-mediated inflammation. The results showed that NOD-stimulatory molecules were secreted by F. nucleatum in the spent culture broth, which subsequently induced cytokine and antimicrobial peptide expression in oral epithelial cells. In the spent broth from T. forsythia-F. nucleatum co-cultures, the NOD-stimulatory activity was significantly reduced. These data indicated that F. nucleatum releases NOD2-stimulatory muropeptides in the environment, and T. forsythia can effectively scavenge the muropeptides released by co-habiting bacteria to dampen NOD-mediated host responses. This proof-of-principle study demonstrated that peptidoglycan scavenging by T. forsythia can impact the innate immunity of oral epithelium by dampening NOD activation.

Keywords: Fusobacterium nucleatum; NOD; Tannerella forsythia; muramyl peptides; peptidoglycan; periodontitis.

Fig.1.. F. nucleatum spent broth supports T. forsythia growth.

Planktonic growth of T. forsythia was monitored in spent broth from the cultures of F. nucleatum (Fn.SB), P. gingivalis (Pg.SB) or S. gordonii (Sg.SB). For this purpose, each test species was grown in BHI broth anaerobically to an optical density of 1.0 at 600 nm. The spent broth was then filtered through a 0.2 μm filter and inoculated with T. forsythia cells to a final O.D. of 0.05. The culture was grown under anaerobic conditions for 7 days with continuous monitoring of growth at 600 nm. Data present means S.D. of triplicate samples. A representative result from two independent experiments is shown. *, p < 0.05 (Fn.SB compared with BHI.MurNAc, Pg.SB and Sg.SB at each time point).

Fig. 2.. F. nucleatum spent medium activates NOD2.

HEK-hNOD2 reporter cells were stimulated for 16 h with bacterial spent broth at a concentration of 5% or 20% of the total cell culture volume. The level of NOD2 activation was determined by assaying for SEAP enzyme activity (OD520 nm). The results shown are mean ± SD of triplicate cultures and representative of three independent experiments. Abbreviations: MDP, muramyl di-peptide (positive control); BHI, brain heart infusion broth (sterile control); Fn.SB, F. nucleatum spent broth; Pg,SB, P. gingivalis spent broth; Sg.SB, S. gordonii spent broth. The small letters above columns indicate significance. The groups labeled with the same letter are not statistically different from each other while the groups labeled with different letters are significantly different from each other (p < 0.05).

Fig. 3.. F. nucleatum secreted peptidoglycan fragments induce NOD-dependent cytokine secretion from oral epithelial cells.

OBA-9 cells were stimulated with F. nucleatum spent broth in the presence or absence of a RIPK2 inhibitor which blocks NOD signaling. The levels of IL-1β and IL-6 in the supernatant were assayed by ELISA. Data are representative of two independent experiments. ***, p < 0.001.

Fig. 4.. T. forsythia scavenges F. nucleatum secreted NOD2 stimulatory molecules.

F. nucleatum and T. forsythia cells were cultured anaerobically in a transwell tissue culture dish with a 0.45 μm filter and after 48 h the spent broth from the T. forsythia well was collected and incubated with HEK-hNOD2 reporter cells (20% spent broth in total cell culture medium) for assaying NOD2 stimulatory activity. As shown the spent broth from the T. forsythia - F. nucleatum co-culture condition (Fn/TfWT) induced significantly less NOD2 activity as compared to the spent broth from the culture of F. nucleatum alone (Fn/-) or the co-culture of F. nucleatum - T. forsythia muropeptide deficient mutant (Fn/TfΔampG). The results shown are mean ± SD of triplicate cultures and representative of three independent experiments. *, p < 0.05; ***, p < 0.001.

Fig. 5.. T. forsythia scavenges NOD ligands to dampen cytokine and antimicrobial human β-defensin secretion from oral epithelial cells.

OBA-9 cells were stimulated with spent broth collected as in the Fig. 4 for 16 h. MDP and sterile broth were used as positive and negative controls, respectively. The levels of cytokines and HBD from the culture supernatants were assayed by ELISA. Data are mean ± SD of two independent experiments each performed with three replicates per condition. *, p < 0.05; ***, p < 0.001.