Open Access
lnsights into bacterial functional amyloids using the staphyiococcal Bap protein asan amyloid model
(2022) Matilla Cuenca, Leticia; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
Los arniloides funcionales son componentes clave de la matriz del biofilrn de muchas bacterias. Estas estructuras proporcionan resistencia a las proteasas y a condiciones adversas de desnaturalización, y refuerzan y dan integridad a la estructura del biofilrn. La proteína Bap (Biofilrn associated protein) de Staphylococcus aureus es una proteína de superficie capaz de formar estructuras de tipo arniloide. Bap es un miembro de la familia BAP que comprende proteínas de alto peso molecular, que contienen un dominio elevado de repeticiones y que desempeñan importantes funciones en la formación de biofilrns y en la virulencia. Cabe destacar que las proteínas BAP están ampliamente distribuidas entre las bacterias Grarn positivas y Grarn negativas del biofilrn formado por la rnicrobiota intestinal. Utilizando Bap de S. aureus como modelo de arniloide funcional, en esta tesis hemos investigado si la formación de biofilrns por ensamblaje de agregados arniloides es un mecanismo extendido entre las proteínas BAP de la rnicrobiota intestinal. En el Capítulo I demostrarnos que Esp, una proteína ortóloga a Bap producida por Enterococcus faecalis, forma agregados de tipo arniloide en condiciones ácidas. Demostrarnos que la región N-terrninal de Esp comprende un elevado número de péptidos arniloides y forma estructuras fibrilares con características arniloides, tal y corno demuestran los análisis de microscopía electrónica, las pruebas biofísicas y la unión de los agregados proteicos a colorantes indicativos de arniloides. La región N-terrninal de Esp es capaz de inducir un comportamiento rnulticelular en bacterias que carecen de Esp, tanto cuando se añade exógenarnente al cultivo corno cuando se expresa a partir de un plásrnido. Más adelante, en el Capítulo II, demostrarnos que las proteínas BAP de la rnicrobiota intestinal contienen dominios con propiedades arniloides. Utilizando el sistema generador de arniloides dependiente de curli (C-DAG) validarnos las propiedades arniloidogénicas de los dominios predichos. Caracterizarnos los dominios arniloidogénicos mediante técnicas microscópicas, bioquímicas y biofísicas para concluir que las proteínas BAP de las bacterias patobiontes y comensales del intestino forman estructuras de tipo arniloide. Por lo tanto, la polimerización de proteínas rnonornéricas en arniloides funcionales parece ser un mecanismo generalizado de las proteínas de la familia BAP de la rnicrobiota intestinal. Con el fin de controlar la polimerización de proteínas arniloides en bacterias patógenas, en el Capítulo III analizarnos las propiedades anti-arniloides de una batería de polifenoles. Identificarnos que los flavonoides quercetina, rniricetina y escutelareína inhiben específicamente el biofilrn mediado por Bap en cepas de S. aureus y en otras especies de estafilococos que expresan Bap. Demostrarnos que los polifenoles no afectan a la expresión de Bap, sino que inhiben el ensamblaje de Bap en estructuras arnioides. Además, la inoculación subcutánea de quercetina y rniricetina inhiben la colonización de una cepa de S. aureus que expresa Bap en catéteres implantados en un modelo modelo rnurino de infección por catéter. Por otro lado, decidirnos utilizar los arniloides y beneficiarnos de sus propiedades para construir biofilrns funcionales. En el Capítulo IV, crearnos biofilrns funcionalizados mediante la ingenierización de la proteína Bap con dominios funcionales. Corno prueba de concepto, decoramos la superficie bacteriana con etiquetas con diferentes funciones que incluyen la unión de iones metálicos, la emisión de fluorescencia, la adhesión y la inmovilización covalente de moléculas. Utilizando estas herramientas, demostrarnos el papel dual de Bap, corno adhesina en la superficie celular o corno componente extracelular de la matriz del biofilrn, mediante la simple modulación del pH del medio. Finalmente, crearnos biofilrns funcionalizados añadiendo exógenarnente un dominio arniloidogénico de Bap ingenierizado. Con este enfoque, somos capaces de inducir la formación del biofilrn de forma heteróloga en bacterias no modificadas genéticamente.
Open Access
Bacterial biofilm functionalization through Bap amyloid engineering
(Springer Nature, 2022) Matilla Cuenca, Leticia; Taglialegna, Agustina; Gil Puig, Carmen; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
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. © 2022, The Author(s).
Open Access
Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids
(Nature Research, 2020) Matilla Cuenca, Leticia; Gil Puig, Carmen; Cuesta Ferre, Sergio; Rapún Araiz, Beatriz; Mira, Alex; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ziemité, Miglé; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, PI011 KILL-BACT
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.
Open Access
The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers
(Nature Research, 2020) Taglialegna, Agustina; Matilla Cuenca, Leticia; Dorado Morales, Pedro; Navarro, Susanna; Ventura, Salvador; Garnett, James A.; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
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.
Open Access
Anti-biofilm molecules targeting functional amyloids
(MDPI, 2021) Matilla Cuenca, Leticia; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
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.
Open Access
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
(MDPI, 2022) Bastet, Laurène; Bustos-Sanmamed, Pilar; Catalán Moreno, Arancha; Caballero Sánchez, Carlos; Cuesta Ferre, Sergio; Matilla Cuenca, Leticia; Villanueva San Martín, Maite; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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.