Mechanistic investigation of multispecies interactions in clear aligner induced periodontal inflammation

Abstract Clear aligner treatment can lead to oral microbial shift which may impact periodontal health. The interplay between microbes and the host plays critical roles in health and diseases. However, a mechanistic understanding of how the shifted microbiome (dysbiosis) and its interaction with the host influence oral health is still lacking. Our preliminary studies demonstrated the increased relative abundance of Fusobacterium nucleatum (Fn), Actinomyces spp. and Saccharibacteria (TM7) positively correlated with the progression of gingivitis in clear aligner patients. Spectral FISH imagining of clinical samples and in vitro co-aggregation analysis indicated a close physical association between Fn, Actinomyces spp., and TM7. More importantly, we provided new preliminary data to demonstrate the potential synergy among the three bacterial species via physical interaction and metabolic cross-feeding. The proposed study focuses on these three species which have a myriad of virulence factors relevant to oral and systemic diseases and positively associated with periodontal inflammatory diseases. These intriguing findings serve as the scientific premise for the working hypothesis that Fn, Actinomyces spp. and TM7 may engage in synergistic interactions to enhance their growth within biofilm and exhibit cooperative effects when interacting with host oral epithelial cells to promote inflammation. The well- studied type strains Fn ATCC 23726, A. odontolyticus XH001 and Nanosynbacter lyticus strain TM7x, which represent Fn, Actinomyces spp., and TM7, respectively, will be used in this study. Two independent but interconnected aims are developed to test the working hypothesis: Aim 1. Investigate the synergism between Fn, A. odontolyticus, and TM7 during biofilm formation. Using Fn 23726, XH001 and TM7x model strains, comprehensive imaging-based analysis will be employed to investigate the synergistic interaction during biofilm growth by assessing biofilm biovolume, maximal thickness, roughness correlation ratio, and continuity ratio via confocal laser scanning microscopy (CLSM). Aim 2. Examine the cooperative effects between Fn, A. odontolyticus, and TM7 when interacting with host oral epithelial cells. We will use an in vitro culture system with various combinations of Fn 23726, XH001 and TM7x wildtype strains in the presence or absence of human oral keratinocytes (HOK-16B) to identify genes with expression changes specifically attributable to the synergy of three species during interactions with HOK- 16B cells and determine if tri-species interaction has cooperative effects contributing to gingival inflammation. The successful completion of these aims will provide novel mechanistic-level insight into the impact of microbial shift during treatment with clear aligners on periodontal health. More importantly, the study will help identify the molecular targets involved in polymicrobial synergy contributing to periodontal disease initiation/progression for potential therapeutic purposes in the future.