Pathobiont-mediated spatial structuring enhances biofilm virulence in childhood oral disease

Abstract Microbiome studies are revealing complex microbiota in biofilm-mediated human diseases commonly linked with specific bacterial pathogens. Streptococcus mutans has been implicated as the primary pathogen in childhood dental caries (tooth decay). While the role of polymicrobial communities is appreciated, it remains unclear whether other microorganisms are active contributors, inactive cohabitants, or interact with known pathogens such as S. mutans. Here, we integrate multi-omics of human dental plaque (biofilm) from two community-based samples of preschool-age children in a discovery-validation pipeline involving bioinformatics, laboratory, and in vivo experimental approaches to identify disease-relevant inter-species interactions. In metagenomics and metatranscriptomics analyses among 416 preschool-age children, we identify 16 taxa strongly associated with childhood caries. Using multiscale imaging, virulence assays, microcalorimetry, and computational analyses, we investigate biofilm formation dynamics, microscale spatial arrangement, and metabolic activity by Selenomonas sputigena, Prevotella salivae and Leptotrichia wadei either individually or with S. mutans. Notably, we discover that the flagellated S. sputigena, a subgingival anaerobe with previously unknown role in supragingival biofilm virulence, becomes trapped in foreign streptococcal exoglucans, loses its motility but actively proliferates to build a honeycomb-like multicellular superstructure encapsulating S. mutans and enhances acidogenesis. Rodent model experiments reveal a previously unrecognized ability of S. sputigena to colonize supragingival tooth surfaces. While incapable of causing caries on its own, S. sputigena exacerbates the disease severity in vivo when co-infected with S. mutans, causing extensive lesions on tooth enamel. Our data reveal a pathobiont from a disparate habitat in a prevalent disease that cooperates with a known pathogen to build a unique 3D spatial structure and heighten biofilm virulence—which may be relevant to other polymicrobial diseases.