Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced byEnterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.

The choice of an effective therapeutic strategy in the treatment of biofilm-related infections is a significant issue. Amyloids, which have been historically related to human diseases, are now considered to be prevailing structural components of the biofilm matrix in a wide range of bacteria. This assumption creates the potential for an exciting research area, in which functional amyloids are considered to be attractive targets for drug development to dissemble biofilm structures. The present review describes the best-characterized bacterial functional amyloids and focuses on anti-biofilm agents that target intrinsic and facultative amyloids. This study provides a better understanding of the different modes of actions of the anti-amyloid molecules to inhibit biofilm formation. This information can be further exploited to improve the therapeutic strategies to combat biofilm-related infections.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TT_sbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TT_sbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TT_sbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TT_sbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

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.