Role of Streptococcus gordonii Cbe protein in periodontopathic biofilm development

Objectives: In the process of dental plaque development, pioneer organisms, such as Streptococcus gordonii, facilitate the colonization of later arrivals including periodontopathic bacteria, Fusobacterium nucleatum and Porphyromonas gingivalis. We previously reported that S. gordonii mutants deficient in Cbe (putative chorismate binding enzyme) and SpxB (pyruvate oxidases), failed to support a heterotypic biofilm community with P. gingivalis. In this study, we investigated the roles of Cbe and SpxB in heterogenic plaque formation by S. gordonii, F. nucleatum and P. gingivalis in an in vitro subgingival biofilm model using confocal laser scanning microscopy.

Methods: Cbe and SpxB deficient mutants of S. gordonii (ΔCBE and ΔSPX, respectively) were used to analyze biovolume and structure of three-species mixed biofilms. S. gordonii cells were stained with hexidium iodide, then cultured anaerobically for 16 h in saliva-coated chambers of a Culture Well^TM chambered coverglass system. F. nucleatum and P. gingivalis cells were stained with FITC and DAPI respectively, and reacted with the S. gordonii biofilm for 24 h each in series/inverted series. Multi-species biofilms developed on the coverglass were observed on a Bio-Rad Radiance2100 CLSM system and fluorescence was quantified by IMARIS Measurement Pro 5.0 software.

Results: Loss of Cbe drastically decreased the biovolume of P. gingivalis within a S. gordonii-F. nucleatum biofilm (p=0.0228), whereas the ΔSPX-F. nucleatum modestly recruited P. gingivalis to the mixed community. Interestingly, F. nucleatum was also diminished in ability to accumulate within the ΔCBE-P. gingivalis mixed biofilms (p=0.0122). Loss of SpxB showed little effect on F. nucleatum accumulation in S. gordonii-P. gingivalis biofilms.

Conclusion: The influence of SpxB deletion in S. gordonii can be complemented by other organisms in dental plaque, whereas the function of Cbe is crucial for maturation of S. gordonii-F. nucleatum-P. gingivalis communities.