Role of pili in S.sanguinis to the cell adhesion

Objectives: Oral bacterial biofilm are associated with an increased risk of atherosclerosis. Streptococcus sanguinis is a member of the viridians group of streptococci and a primary colonizer of the human oral cavity. Pili of S. sanguinis are reported to be involved in adhesion to human cells. In our previous study, we developed a micro-channel chip to observe the cell adhesion process in a fluid system. The aim of this study is to investigate the role of pili in S. sanguinis–induced plaque adhesion in this system. Methods: The flow test on the micro-channel chip was conducted to clarify that S. sanguinis strains are related to plaque-like formation of murine macrophage RAW264.7 cells. At first, we constructed the fluorescence strain using plasmid expressed green fluorescent protein for visualization of S. sanguinis. We constructed ldh promoter-gfp fragment into pDL278 plasmid as a shuttle vector. pDL278-gfp was transformed to S. sanguinis SK36 and S. sanguinis SK36 pili-/-.These strains were grown in Brain Heart Infusion (BHI) with selection for spectinomycin (spe:1000 µg/ml) supplement with sucrose (final concentration of 0.5%) medium. We also confirmed that these strains expressed green fluorescence protein using a fluorescence microscope. The RAW264.7 cells were cultured in the presence or absence of 1 µg/ml LPS for 12 h. Prior to the flow test, cells were prepared at a concentration of 1.5×10⁶ cells/ml. Results: We detected fluorescence in S. sanguinis SK36/pDL278-gfp using a fluorescence microscope. S. sanguinis SK36/pDL278-gfp adhered to plaque-like formation of LPS-stimulated macrophages on the micro-pillars of the micro-channel chip. Conclusion: These results suggest that the pili in S. sanguinis play an important role in the adhesion on plaque-like formation of LPS-stimulated macrophages.