The interaction of F. nucleatum and P. gingivalis appeared to be mediated by an adhesion protein identified as the outer membrane protein FomA on F. nucleatum and a carbohydrate receptor on P. gingivalis [18] and [33], although only a few studies have shown a role for FomA in the pathogenesis of periodontal diseases and halitosis [25]. Our data demonstrate for buy MI-773 the first time that F. nucleatum co-opts P. gingivalis via FomA to enhance co-aggregation, biofilm formation, gum inflammation, and VSC production. Co-aggregation

between F. nucleatum and P. gingivalis strains has been previously observed using either a macroscopic visual co-aggregation assay, based on radioactive labeling of bacteria, or using fluorochromes

and confocal microscopy [32]. Although assaying co-aggregation by detecting visible clusters of bacteria is a common method, one main disadvantage of the method is the inability to dynamically quantify the co-aggregation. This method also lacks the capability of verifying the physical interactions among bacteria although bacterial clusters can be observed. On the other hand, the use of Malvern Zetasizer Nano-ZS equipped with DLS provides the ability to detect an increase in particle sizes derived from the physical aggregation of multiple particles [32]. Although F. nucleatum is a spindle-shaped bacterium, a size distribution between 342 and 712 nm is detected by the DLS analysis of Malvern Zetasizer Nano-ZS. Size analysis of the co-aggregation of F. nucleatum and P. gingivalis using Malvern Zetasizer Nano-ZS showed the presence

of larger Kinase Inhibitor Library mouse aggregates (712–1281 nm) ( Fig. 1B), verifying the physical interaction between two bacteria. Although we observed larger aggregates in the co-culture of bacteria on nonpyrogenic polystyrene plates ( Fig. 1A), these larger aggregates were undetectable by Malvern Zetasizer Nano-ZS. Possible explanations include that the Malvern Zetasizer Nano-ZS has a limitation that restricts its ability to detect particle sizes greater than 6000 nm. It is also possible that bacteria on the nonpyrogenic polystyrene plates formed larger aggregates L-NAME HCl than those bacteria suspended in the bacterial medium during Malvern Zetasizer Nano-ZS analysis. It is worthwhile to note that only few P. gingivalis (103 CFU) are needed to trigger the enhancement of bacterial co-aggregation between F. nucelatum (4 × 108 CFU) and P. gingivalis ( Supplementary Fig. 1). This result is consistent with recent findings that a low dose of P. gingivalis (106 CFU) synergistically enhances the pathogenicity of F. nucleatum (109 CFU) in a murine model using subcutaneously implanted chambers [32] and [34]. Thus, besides the physical interaction among bacteria, bacterial co-aggregation may also be strengthened by quorum sensing mechanisms [35].