Dental caries, commonly known as tooth decay or cavities, remains one of the most common and costly infectious diseases worldwide. According to the CDC, associated health care costs tens of billions of dollars annually, and the rates of childhood caries in the United States are rising. Novel, comprehensive strategies are needed to effectively combat caries pathogenesis. Cariogenic bacteria form tenacious biofilms on the surface of teeth known as dental plaque. Extracellular deoxyribonucleic acid (eDNA) is an integral component of the extracellular polymeric matrix (ECM) of biofilms. Novel approaches to target the ECM to impede biofilm development and persistence are promising routes to prevent and treat biofilm- associated diseases. eDNA is recently recognized to play a pivotal role in bacterial biofilm formation, yet how eDNA mediates surface and intercellular adherence and biofilm accumulation, structure and stability in S. mutans or other organisms has not been extensively studied. We have evidence that S. mutans actively releases eDNA via production of membrane vesicles (MVs). The existence of MVs is well documented in Gram-negative bacteria but only recently discovered in Gram-positive bacteria. Production of eDNA is up- regulated in early biofilms and destruction of this eDNA significantly reduces biofilm formation and weakens biofilm stability. Deficiency of sortase A (SrtA), a transpeptidase known to covalently link multiple surface- associated proteins to the peptidoglycan, does not impact MV production, however substantially influences their protein composition including those known to contribute to S. mutans biofilm formation and cariogenicity. In contrast to wild-type S. mutans, the srtA mutant does not build eDNA nanofibers, even though MVs from the mutant still contain eDNA. In this exploratory R21, we will (i) use electron microscopy (EM) and immunogold EM techniques to explore the biogenesis of S. mutans MVs and confirm the MV- eDNA relationship, (ii) test the hypothesis that MV biogenesis and eDNA release are genetically regulated and environmentally influenced processes, and (iii) evaluate the contribution of individual SrtA substrate proteins to the structure and function of eDNA nanofibers in biofilms and assess their cooperative effects on eDNA-mediated biofilm formation. These studies will verify that bacteria use MVs as a vehicle for active eDNA release and will provide novel insights into vesiculogenesis by S. mutans and other Gram-positive bacteria. Importantly, identification of factor(s) modulating active release of eDNA via MVs, and subsequent nanofiber formation and functional activity of eDNA will improve our understanding of the complex process of biofilm formation by S. mutans and thereby provide insight for the development of novel, comprehensive strategies to combat tooth decay as well as other biofilm-associated diseases.