Amyloid structures as biofilm matrix scaffolds

Recent insights into bacterial biofilm matrix structures have induced a paradigm shift toward the recognition of amyloid fibers as common building block structures that confer stability to the exopolysaccharide matrix. Here we describe the functional amyloid systems related to biofilm matrix formation in both Gram-negative and Gram-positive bacteria and recent knowledge regarding the interaction of amyloids with other biofilm matrix components such as extracellular DNA (eDNA) and the host immune system. In addition, we summarize the efforts to identify compounds that target amyloid fibers for therapeutic purposes and recent developments that take advantage of the amyloid structure to engineer amyloid fibers of bacterial biofilm matrices for biotechnological applications.This work, including the efforts of Jaione Valle, was funded by Ministerio de Economía y Competitividad (MINECO) (AGL2011-23954). This work, including the efforts of Íñigo Lasa, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and BIO2014-53530-R)

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. © 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

Estudio funcional y estructural de la región B de Bap y su rol en el desarrollo de biofilms en S.aureus

Trabajo presentado en la X Reunión de Microbiología Molecular, celebrada en Segovia del 9 al 11 de junio de 2014.Peer Reviewe

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.This research was supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from the Spanish Ministry of Science, Innovation and Universities, and Proyecto Intramural Incorporación-2018 CSIC

Funcionalización de biofilms mediante la ingenierización de la proteína amilode Bap

Resumen del trabajo presentado en el XXVIII Congreso Nacional de la Sociedad Española de Microbiología, celebrado de forma virtual del 28 de junio al 2 de julio de 2021Las fibras amiloides son un elemento estructural común de la matriz extracelular del biofilm en diversas bacterias. La capacidad de autoensamblaje de las proteínas amiloidogénicas, junto con la estabilidad y robustez de las nanofibras que generan, convierten a los amiloides en complejos proteicos con excepcionales cualidades para la construcción de filamentos funcionales a escala nanométrica con posibles propiedades terapéuticas. Los amiloides facultativos tienen además la ventaja de desempeñar una doble función. La proteína en su estado nativo puede actuar como adhesina, y en la conformación amiloide como andamiaje de la matriz extracelular del biofilm. Utilizando como modelo de amiloide facultativo la proteína Bap de Staphylococcus aureus, hemos ingenierizado la región amiloide N-terminal de Bap con diferentes dominios funcionales. La expresión de estas proteínas ha dado lugar a diversas nanoestructuras funcionalizadas que, en función de las condiciones ambientales, están asociadas a la superficie de las células o como parte de la matriz extracelular del biofilm. La segunda estrategia para funcionalizar fibras amiloides ha consistido en la complementación exógena de bacterias con el dominio amiloide N-terminal de Bap recombinante funcionalizado con diferentes etiquetas. La ventaja de este sistema es que las cepas derivadas de la complementación exógena no contienen material genético modificado. Estas estrategias nos permiten crear por primera vez amiloides facultativos funcionales y modificables que pueden ser empleados como herramientas biotecnológica

VraSR and Virulence Trait Modulation during Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus Infection

Methicillin-resistant S. aureus continues to develop resistance to antimicrobials, including those in current clinical use as daptomycin (DAP). Resistance to DAP arises by mutations in cell membrane and cell wall genes and/or upregulation of the two-component VraSR system. However, less is known about the connection between the pathogen and virulence traits during DAP resistance development. We provide new insights into VraSR and its regulatory role for virulence factors during DAP resistance, highlighting coordinated interactions that favor the higher persistence of MRSA DAP-resistant strains in the infected host.Methicillin-resistant Staphylococcus aureus (MRSA) threatens human health in hospital and community settings. The lipopeptide antibiotic daptomycin (DAP) is a frequently used treatment option for MRSA infection. DAP exposure can cause bacterial resistance because mutations are induced in genes implicated in cell membrane and cell wall metabolism. Adaptations aimed at surviving antimicrobial pressure can affect bacterial physiology and modify in vivo aptitude and pathogenesis. In this study, clinical DAP-susceptible (DAPs) and DAP-resistant (DAPr) MRSA isolates were used to investigate associations between DAP resistance and staphylococcal virulence. We previously found that VraSR is a critical sensor of cell membrane/wall homeostasis associated with DAP acquisition during MRSA infection. The present study found that DAPr CB1634 and CB5014 MRSA strains with vraSR upregulation were less virulent than their susceptible counterparts, CB1631 and CB5013. Differential gene-transcription profile analysis revealed that DAPr CB1634 had decreased agr two-component system expression, virulence factors, and highly suppressed hemolysis activity. Functional genetic analysis performed in DAPr CB1634 strains using vraSR inactivation followed by gene complementation found that vraSR acted as a transcriptional agrA regulator. These results indicated that VraSR has a broad range of regulatory functions. VraSR also appeared to affect DAPr adherence to epithelial cells, which would affect DAPr strain colonization and survival in the host. The correlation between DAP resistance and decreased virulence was also found in the CB5013 (DAPs) and CB5014 (DAPr) pair. Taken together, these findings are the first evidence that DAP resistance and MRSA virulence are tightly connected and involve compromised expression of regulatory and virulence determinants

Estudio funcional de la proteína Bap en las interacciones bacteria-bacteria en Staphylococcus aureus

Trabajo presentado en la IX Reunión del Grupo de Microbiología Molecular, celebrada en Palma de Mallorca del 14 al 16 de noviembre de 2012.El desarrollo de comunidades de bacterias que crecen embebidas en matrices extracelulares y adheridas a superficies se denomina biofilm. La familia de proteínas BAP (Biofilms-Associated Protein) parece constituir uno de los elementos comunes al proceso de formación de la matriz del biofilm en muy diversas especies bacterianas. Sin embargo, los mecanismos moleculares mediante los cuales las proteínas BAP promueven las interacciones entre las bacterias son desconocidos. En este trabajo hemos utilizado la proteína Bap de Staphylococcus aureus como modelo para investigar los mecanismos moleculares mediante los cuales esta proteína promueve el establecimiento de interacciones intercelulares. Utilizando técnicas de microscopia electrónica e inmunifluorescencia se determinó que Bap se localiza en la superficie de S. aureus donde se acumula preferentemente en los polos viejos de división. Ensayos de cinética de agregación e inhibición del biofilm con anticuerpos específicos contra Bap, moestraron que Bap cinética dea gregación e inhibición del biofilm con anticuerpos específicos contra Bap, mostraron que Bap juega un papel importante en la interacción entre bacterias favoreciendo la formación de biolfilm. La expresión de Bap en cepas de S. aureus Bap-negativas promovió la formación de biofilm. Sin embargo, la complementación heteróloga de otros géneros bacterianos, no dió lugar a un claro fenotipo de formación de biofilm. Finalmente, las técnicas de electroforesis de proteínas en geles nativos y crosslinking in-vivo mostraron que Bap es capaz de multimerizar independiente de la presencia de calcio en el medio de cultivo. Los dominios coiled-coil presentes en la región A de la proteína Bap parece que están implicados en formación de los oligomeros intermedios de Bap. Para concluir, todos estos resultados demuestran que Bap juega un papel esencial en la formación del biofilm de S. aureus, al promover la agregación intercelular mediante determinadas regiones que podrían incluir los dominios coiled-coil. Se están llevando a cabo más estudios para identificar otras regiones de la proteína implicadas en la interacción entre proteínas Bap.Peer Reviewe

Functional characterization of Bap subdomains required for biofilm development and host cell interaction in S. aureus

Trabajo presentado en el 60th Nobel Conference on Biofilm Formation, its Clinical Impact and Potential Treatment, celebrado en Estocolmo del 28 al 30 de agosto de 2013.Bap is the prototype of the proteinaceous biofilm matrix in bacteria. Members of this family confer the bacteria the capacity to form a biofilm and to interact with host cells. As common structural features, Bap-­‐related proteins: (i) show a high molecular weight; (ii) contain a core domain of tandem repeats and (iii) very often are harboured in mobile genetic elements. Most of our knowledge about the functionality of Bap proteins has been obtained with the Bap protein of S. aureus. This protein is 2276-­‐amino acid long and display the multidomain architecture characteristic of surface-­‐associated proteins from Gram-­‐positive bacteria. Bap protein promotes biofilm development on abiotic surfaces as well as intercellular adhesion in a process that it is inhibited by the presence of Ca+2. Bap also promotes the adhesion to epithelial cells but inhibits epithelial cell invasion through the interaction with host receptor Gp96 (Valle et al., Plos Pathog 2012). Several evidences suggest that Bap-­‐Gp96 interaction interferes with the fibronectin-­‐binding protein mediated invasion pathway. Although the role of Bap in biofilm development and host cell interaction is well established, our understanding of the Bap domains responsible for these phenotypes remains poorly characterized. In this communication we will present a functional characterization of the Bap subdomains necessary to promote multicellular behavior under in vitro conditions and bacterial adhesion to eukaryotic cells. By using chimeric proteins containing different regions of the Bap protein fused with the clumping factor subdomain R, we have determined the minimal region of Bap necessary and sufficient to mediate biofilm formation and cell-­‐to-­‐cell interactions. Interestingly, this subdomain contains the EF-­‐hand motifs and bacteria producing this subdomain are sensitive to the presence of Ca+2. We have also identified the region of Bap that it is responsible for the interaction with Gp96. We will discuss how the identification of these two subdomains will allow us to evaluate the relative contribution of biofilm development and host cell interaction during Bap-­‐mediated S. aureus chronic infection.Trabajo presentado en la 60th Nobel Conference on Biofilm Formation, its Clinical Impact and Potential Treatment, celebrada en Suecia en 2013.Peer Reviewe

Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria