Gut microbiota produces biofilm-associated amyloids with potential for neurodegeneration.

Age-related neurodegenerative diseases involving amyloid aggregation remain one of the biggest challenges of modern medicine. Alterations in the gastrointestinal microbiome play an active role in the aetiology of neurological disorders. Here, we dissect the amyloidogenic properties of biofilm-associated proteins (BAPs) of the gut microbiota and their implications for synucleinopathies. We demonstrate that BAPs are naturally assembled as amyloid-like fibrils in insoluble fractions isolated from the human gut microbiota. We show that BAP genes are part of the accessory genomes, revealing microbiome variability. Remarkably, the abundance of certain BAP genes in the gut microbiome is correlated with Parkinson's disease (PD) incidence. Using cultured dopaminergic neurons and Caenorhabditis elegans models, we report that BAP-derived amyloids induce α-synuclein aggregation. Our results show that the chaperone-mediated autophagy is compromised by BAP amyloids. Indeed, inoculation of BAP fibrils into the brains of wild-type mice promote key pathological features of PD. Therefore, our findings establish the use of BAP amyloids as potential targets and biomarkers of α-synucleinopathies.

Bacterial biofilm functionalization through Bap amyloid engineering.

Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

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 (TTsbrB) 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 TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB 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 TTsbrB, 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.

Anti-Biofilm Molecules Targeting Functional Amyloids.

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.

Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids.

The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The Biofilm Associated Protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.

The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers.

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 by Enterococcus 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.