Biofilm formation on collagen substrates modulates <i>Streptococcus mutans</i> bacterial extracellular nanovesicle production and cargo

<i>Streptococcus mutans</i> is the major microbial etiological agent of dental caries and can adhere to surfaces such as type-I collagen, which is present in dentin and periodontal tissues. Recent studies have characterized planktonic <i>S. mutans</i> bacterial extracellular vesicles (bEVs) at the nanoscale range and demonstrated environmental-induced changes due to sugar presence or pH alterations. However, to date, no studies have explored whether surface-derived changes can modulate bEV production in the context of oral biofilm formation in the elderly. Therefore, this work aimed to determine the role of biofilm formation and collagen glycation on the nanoscale morphology and proteomic composition of <i>S. mutans</i> bEVs. For this, bEVs from <i>S. mutans</i> biofilms on native and glycated collagen surfaces were isolated, characterized, and compared to bEVs from planktonic cells. Nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), and electron microscopy confirmed bEV production and showed that bEVs from biofilms are smaller in size and less abundant than those from planktonic cells. Furthermore, proteome analysis revealed that <i>S. mutans</i> biofilm formation on native and glycated collagen led to the enrichment of several key virulence proteins. Also, a shift towards proteins involved in metabolic processes was found in bEVs following biofilm formation on collagen surfaces, whereas glucan metabolism proteins were overexpressed in vesicles from the planktonic state. These results demonstrate that biofilm formation, as well as the glycation of collagen associated with aging and hyperglycaemia, can modulate bEV characteristics and cargo and could play a central role in <i>S. mutans</i> virulence and the development of diseases such as dental caries and periodontal disease.