BUSCADOR RECOLECTA

El tutor de la tesis es Íñigo Lasa Uzcudun, 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., 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., Este trabajo ha sido realizado dentro de los siguientes proyectos de investigación: 1. Caracterización de los arniloides funcionales tipo BAP del rnicrobiorna intestinal y evaluación de su papel en el desarrollo de enfermedades neurodegenerativas. RTI2018-096011-B-IOO MINECO. Duración: 2019-2021. 2. Estudio de la regulación de la expresión de las proteínas de la familia BAP y análisis de sus propiedades arniloides. Proyecto Intrarnural CSIC. Duración: 2018-2019. 3. Estudio de la formación de fibras miloides como estructura para organizar la matriz de los biofilms bacterianos. SAF2015-74267-JIN MINECO. Duración: 2017-2019, Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

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)., This research was supported by grants from the Spanish Ministry of Science and Technology RTI2018-096011-B-I00 to J.V. and PID2020-113494RB-I00 to IL. L.M.-C. was supported by the predoctoral program of the Universidad Pública de Navarra.

Incluye material complementario, 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., This research was supported by Grants RTI2018-096011-B-I00 from Ministry of Science and Innovation and PI011 KILL-BACT from the Government of Navarra.