Functional amyloid formation by Streptococcus mutans.

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe.