DESCIFRANDO LAS SINGULARIDADES DEL EXOPOLISACARIDO UNIVERSAL DEL BIOFILM (PNAG) Y EVALUACION DE SU POTENCIAL BIOTECNOLOGICO

BIO2014-53530-R

Nombre agencia financiadora Ministerio de Economía y Competitividad
Acrónimo agencia financiadora MINECO
Programa Programa Estatal de I+D+I Orientada a los Retos de la Sociedad
Subprograma Todos los retos
Convocatoria Retos Investigación: Proyectos de I+D+I (2014)
Año convocatoria 2014
Unidad de gestión Dirección General de Investigación Científica y Técnica
Centro beneficiario UNIVERSIDAD PÚBLICA DE NAVARRA (UPNA)
Centro realización INSTITUTO DE AGROBIOTECNOLOGIA Y RECURSOS NATURALES (IARN)
Identificador persistente http://dx.doi.org/10.13039/501100003329

Publicaciones

Found(s) 14 result(s)
Found(s) 2 page(s)

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

Dadun. Depósito Académico Digital de la Universidad de Navarra
  • Caballero, C.J. (Carlos J.)
  • Menendez-Gil, P. (Pilar)
  • Catalan-Moreno, A. (Arancha)
  • Vergara-Irigaray, M. (Marta)
  • García, B. (Begoña)
  • Segura, V. (Víctor)
  • Irurzun, N. (Naiara)
  • Villanueva, M. (Maite)
  • Ruiz-de-los-Mozos, I. (Igor)
  • Solano, C. (Cristina)
  • Lasa, I. (Íñigo)
RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.




A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, a-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms

Zaguán. Repositorio Digital de la Universidad de Zaragoza
  • Mahía, A.
  • Conde-Giménez, M.
  • Salillas, S.
  • Pallarés, I.
  • Galano-Frutos, J.J.
  • Lasa, Í.
  • Ventura, S.
  • Díaz de Villegas, M.D.
  • Gálvez, J.A.
  • Sancho, J.
Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1–42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1–42, a-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them.




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

Dipòsit Digital de Documents de la UAB
  • Taglialegna, Agustina|||0000-0003-4844-8720
  • Navarro, Susanna|||0000-0001-8160-9536
  • Ventura, Salvador|||0000-0002-9652-6351
  • Garnett, James A.
  • Matthews, Steve
  • Penades, José R.
  • Lasa, Iñigo|||0000-0002-6625-9221
  • Valle, Jaione|||0000-0003-3115-0207
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.




Amyloid structures as biofilm matrix scaffolds

Digital.CSIC. Repositorio Institucional del CSIC
  • Taglialegna, Agustina
  • Lasa, Íñigo
  • Valle Turrillas, Jaione
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 Lasa Iñigo, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and BIO2014-53530-R, Peer Reviewed




Evaluation of a Salmonella strain lacking the secondary messenger C-di-GMP and RpoS as a live oral vaccine

Digital.CSIC. Repositorio Institucional del CSIC
  • Latasa Osta, Cristina
  • Echeverz, Maite
  • García, Begoña
  • Gil, Carmen
  • García Ona, Enrique
  • Burgui, Saioa
  • Casares, Noelia
  • Hervás-Stubbs, Sandra
  • Lasarte, Juan José
  • Lasa, Íñigo
  • Solano Goñi, Cristina
Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi- GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine., SB is a predoctoral fellow from the Public University of Navarra. CG and BG are recipients of a postdoctoral contract under Grants IIM 13329.RI1 and BIO2011-30503-C02-02, respectively. This work was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2011-30503-C02-02 and BIO2014-53530-R from the Spanish Ministry of Economy and Competitiveness., Peer Reviewed




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

Digital.CSIC. Repositorio Institucional del CSIC
  • Taglialegna, Agustina
  • Navarro, Susanna
  • Ventura, Salvador
  • Garnett, James A.
  • Matthews, Steve
  • Penadés, José R.
  • Lasa, Íñigo
  • Valle Turrillas, Jaione
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., This research was supported by the Spanish Ministry of Economy and Competitiveness grants AGL2011-23954, BIO2014-53530-R and BFU2013-44763-P. JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness., Peer Reviewed




Sensory deprivation in Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Villanueva, Maite
  • García, Begoña
  • Valle Turrillas, Jaione
  • Rapún-Araiz, Beatriz
  • Ruiz de los Mozos, Igor
  • Solano Goñi, Cristina
  • Martí, Miguel
  • Penadés, José R.
  • Toledo-Arana, Alejandro
  • Lasa, Íñigo
12 Páginas, 7 Figuras. Contiene información suplementaria en: http://dx.doi.org/10.1038/s41467-018-02949-y, Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches., This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, BIO2014-53530-R, SAF2014-56716-REDT
, and RTC-2015-3184-1. J.V. was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness., Peer reviewed




A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aβ1–42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms

Digital.CSIC. Repositorio Institucional del CSIC
  • Mahía Moros, Alejandro
  • Conde-Giménez, María
  • Salillas, Sandra
  • Pallarés, Irantzu
  • Galano-Frutos, Juan J.
  • Lasa, Íñigo
  • Ventura, Salvador
  • Díaz de Villegas, María D.
  • Gálvez, José A.
  • Sancho, Javier
Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them., IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SV is supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government., Peer reviewed




The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Caballero Sánchez, Carlos José
  • Menéndez Gil, Pilar
  • Catalán Moreno, Arancha
  • Vergara-Irigaray, Marta
  • García, Begoña
  • Segura, Víctor
  • Irurzun, Naiara
  • Villanueva, Maite
  • Ruiz de los Mozos, Igor
  • Solano Goñi, Cristina
  • Lasa, Íñigo
  • Toledo-Arana, Alejandro
RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5′UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5′UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist., European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [646869]., Peer reviewed




Noncontiguous operon is a genetic organization for coordinating bacterial gene expression

Digital.CSIC. Repositorio Institucional del CSIC
  • Sáenz, Sonia
  • Bitarte, Nerea
  • García, Begoña
  • Burgui, Saioa
  • Vergara-Irigaray, Marta
  • Valle Turrillas, Jaione
  • Solano Goñi, Cristina
  • Toledo-Arana, Alejandro
  • Lasa, Íñigo
Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC-ytkD-MW1731 genes are transcribed as a single transcription unit, whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and
Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia
Española de Investigación/Fondo Europeo de Desarrollo Regional, European
Union). A.T.-A. is supported by the European Research Council under the
European Union’s Horizon 2020 research and innovation programme Grant
Agreement 646869., Peer reviewed