Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy

Abstract

The P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcus mutans is a cell surface-localized protein involved in sucrose-independent adhesion and colonization of the tooth surface. The immunoreactive and adhesive properties of S. mutans suggest an unusual functional quaternary ultrastructure comprised of intact P1 covalently attached to the cell wall and interacting with non-covalently associated proteolytic fragments thereof, particularly the ~57-kDa C-terminal fragment C123 previously identified as Antigen II. S. mutans is capable of amyloid formation when grown in a biofilm and P1 is among its amyloidogenic proteins. The C123 fragment of P1 readily forms amyloid fibers in vitro suggesting it may play a role in the formation of functional amyloid during biofilm development. Using wild-type and P1-deficient strains of S. mutans, we demonstrate that solid state NMR (ssNMR) spectroscopy can be used to 1) globally characterize cell walls isolated from a Gram-positive bacterium and 2) characterize the specific binding of heterologously expressed, isotopically-enriched C123 to cell wall-anchored P1. Our results lay the groundwork for future high-resolution characterization of the C123/P1 ultrastructure and subsequent steps in biofilm formation via ssNMR spectroscopy, and they support an emerging model of S. mutans colonization whereby quaternary P1-C123 interactions confer adhesive properties important to binding to immobilized human salivary agglutinin.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcus mutans adhesin P1 by NMR spectroscopy

“…Previous work had demonstrated the ability of the C123 truncation fragment (aka AgII) of S. mutans P1 to interact with covalently attached full‐length P1 (aka Ag I/II) on the bacterial cell surface , to adhere to salivary agglutinin , and to form amyloid fibrils within biofilms . However, previous experiments lacked sufficient resolution to characterize the molecular nature of the C123 interaction with other P1 polypeptides.…”

The Role of Functional Amyloids in Bacterial Virulence

“…Intact P1 covalently attached to the cell wall, but interacts with more loosely associated proteolytic P1 fragments, particularly the C-terminal fragment C123 originally identified as AgII, to form the functional adhesive layer [136] , [137] , [138] . The C123 fragment of P1 readily forms fiber-like structures in vitro [138] ( Fig. 3 ), suggesting that it may contribute to the formation of functional amyloid during biofilm development.…”

“…These proteins can form amyloid fibers as a result of proteolytic processing or environmental triggers such as pH and divalent ion concentrations. For TasA, TapA, Bap and P1, the resulting fibers have been shown or suggested to serve as ECM component during biofilm formation [138] , [154] , [183] . For others, it is not clear whether the amyloid conformation represents a functional state.…”

Amyloids of multiple species: are they helpful in survival?

“…These bacteria can form biofilms and produce acids from various sugars. Studies identified an amyloid‐forming cell surface protein, adhesin P1, which assists these bacteria in forming biofilms and dental caries when colonizing dental surfaces (Oli et al, ; Tang et al, ). Staphylococcus aureus forms multicellular aggregates and biofilms while colonizing body parts as a commensal.…”