The proposed NIDCR Dentist Scientist Pathway to Independence Award (K99/R00) will provide me advanced research and academic training to become an independent dentist-scientist at the interface of oral microbiology, biophysics, and spatial omics with impact in Early Childhood Caries (ECC). ECC is a major public health problem characterized by high microbial carriage forming intractable plaque-biofilms on teeth exposed to sugar-laden dietary habits. The disease leads to rampant tooth-decay, is costly to treat and can cause systemic complications in children. Previous studies and a recent multi-omics analysis of dental plaque from two large community-based cohorts of pre-school children (>400) have identified Selenomonas sputigena (Ss), a motile bacterium, to be strongly associated with ECC. This finding was further validated in an in vivo caries model whereby Ss exacerbated the severity of carious lesions when co-infected with Streptococcus mutans (Sm). However, the role of Ss and its motility on biofilm formation, interspecies interaction with Sm, and cariogenic functions are unknown. To address this, I will focus on the overall hypothesis that the motile Ss colonizes tooth surface and interacts with Sm to mediate biofilm spatial structuring and community functions that promote emergent caries- causing properties of supragingival biofilms through three Aims: (1) Characterize Ss motility on surface colonization and biofilm initiation; (2) Determine the dynamics of biofilm assembly, spatial transcriptomics and disease-associated functions; (3) Investigate Ss-mediated colonization, interspecies spatial structuring/omics and biofilm virulence in vivo. The outcome will elucidate the role of Ss and its motility in supragingival biofilm formation and Ss contributions to the etiopathogenesis of ECC. During the K99 mentored phase, I will conduct research in the lab of my primary mentor/co-mentor, while developing key expertise in 3 areas: 1) advanced skills in biophysical methods to study motile bacteria; 2) acquiring knowledge and technical skills in single-cell RNA sequencing and spatial transcriptomics; 3) improving scientific communication and grant writing skills. In addition, I will incorporate mentoring and laboratory managing skills as well as networking. In transition to independent R00 phase, I will implement and complete the spatial transcriptomics studies to understand how Ss influence biofilm interspecies interactions and determine its pathogenic role in dental caries in vivo. The data will provide ample opportunities for further mechanistic studies and targeted strategies for ECC. In addition, it will provide a platform to study other motile oral bacteria in health and disease, which remain understudied. Collectively, the proposal will broaden my vision and skills by capitalizing on highly experienced mentor and co- mentor with an interdisciplinary advisory committee with complementary expertise in biophysics, spatial multi- omics, oral microbiome, and clinical research. I will gain essential knowledge, skills, and experience to build my own research program with the goal of receiving an R01 prior to the end of award, and successfully guide me to pursue independent dentist-scientist career in oral microbiology and cariology with multidisciplinary vision.

PROJECT NARRATIVE Early childhood caries (ECC) is the most common pediatric disease worldwide and a major public health problem in the US. We found that Selenomonas sputigena is strongly associated with ECC and enhance caries severity in vivo, but its role on the disease etiopathogenesis is unclear. This study aims to elucidate how this motile microbe develop biofilms using biophysics/spatial-omics methods and mediate virulence to cause severe caries.