lnsights into bacterial functional amyloids using the staphyiococcal Bap protein asan amyloid model
Authors:   , Matilla Cuenca Leticia 
Published: October 2022

Functional arnyloids are key cornponents of the biofilrn rnatrix of many bacteria. These structures provide resistance toward proteases and harsh denaturing conditions, and strengthening against rnechanical or degradative disruption. The Biofilrn associated protein Bap of Staphylococcus aureus is a surface protein able to forrn arnyloid-like structures as biofilrn rnatrix scaffolds. Bap is a rnernber of the BAP farnily, which cornprises high molecular weight proteins containing a core dornain of tandern repeats that play irnportant roles in biofilrn forrnation and virulence. Of interest, BAP proteins are widely distributed arnong Grarn positive and Grarn negative bacteria frorn the enteric biofilrn. Using Bap as a rnodel of functional arnyloid we sougth to investigate whether the rnechanisrn of arnyloid-like aggregation to build the biofilrn rnatrix is widespread arnong BAP proteins frorn gut rnicrobiota. Herein, in Chapter I, we dernonstrated that Esp, a Bap-orthologous protein produced by Enterococcus faecalis forrns arnyloid-like aggregates under acidic conditions. We showed that the N-terrninal region of Esp cornprises a large nurnber of arnyloid peptides and forrns fibrillar structures with arnyloidogenic features, as evidenced by electron rnicroscopy biophysical analysis and the binding of protein aggregates to arnyloid-indicative dyes. The N-terrninal region of Esp is able to induce rnulticellular behavior in bacteria lacking Esp through the forrnation of arnyloid aggregates, both when it is added exogenously to the culture and when it is expressed frorn a plasrnid. Going further, in Chapter II, we dernostrated that BAP-like proteins frorn the gut rnicrobiota cornprise dornains with arnyloid-like properties. We used the curli-dependent arnyloid generator (C-DAG) systern to validate the arnyloidogenic properties of the predicted dornains. We characterise the arnyloidogenic stretches through biochernical and biophysical approaches to conclude that BAP proteins frorn pathobiont and cornrnensal bacteria frorn the gut forrn arnyloid-like structures. Therefore, the rnechanisrn of arnyloid-like aggregation to build the enteric biofilrn rnatrix seerns to be widespread arnong BAP-like proteins. In order to control amyloid polymerisation of pathogenic bacteria, in Chapter III, we analysed the anti-amyloid properties of a battery of polyphenols. We identified flavonoids such as quercetin, myricetin and scutellarein that specifically inhibit Bap mediated biofilms formed by S. aureus and other staphylococcal species expressing Bap. W e show that polyphenols do not affect Bap expression, but inhibit Bap assembly into amyloid structures. Subcutaneous inoculation of quercetin and myricetin inhibits the colonisation of S. aureus of implanted catheters in vivo. In the other direction, we decided to use amyloids and to benefit from their properties to construct tunable biofilms. In Chapter IV, we created functionalised biofilms via the engineering of Bap amyloid with functional domains. As proof of concept, we decorated the bacteria! surface with peptide/protein tags with different functions including metal ions binding, fluorescence emission, adhesion and covalent immobilisation of molecules. We demonstrated the dual role of Bap as a cell surface adhesin and scaffold component of the biofilm matrix by simply modulating the pH of the media without affecting functional domains capabilities. Finally, we created functionalised biofilms by the exogenous addition of the amyloidogenic region of Bap (region B). With this approach, we were able to induce biofilm formation heterologously in non-genetically modified bacteria, solving the current legislation on the use of engineered bacteria.