CARACTERIZACION FUNCIONAL DE LOS DETERMINANTES MOLECULARES PARA LA ADAPTACION DE STAPHYLOCOCCUS AUREUS A LA VIRULENCIA

BIO2017-83035-R

Nombre agencia financiadora Agencia Estatal de Investigación
Acrónimo agencia financiadora AEI
Programa Programa Estatal de I+D+i Orientada a los Retos de la Sociedad
Subprograma Programa Estatal de I+D+i Orientada a los Retos de la Sociedad
Convocatoria Retos Investigación: Proyectos I+D+i
Año convocatoria 2017
Unidad de gestión Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016
Centro beneficiario UNIVERSIDAD PUBLICA DE NAVARRA
Identificador persistente http://dx.doi.org/10.13039/501100011033

Publicaciones

Found(s) 11 result(s)
Found(s) 2 page(s)

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

Dipòsit Digital de Documents de la UAB
  • Taglialegna, Agustina|||0000-0003-4844-8720
  • Matilla-Cuenca, Leticia|||0000-0003-1822-0151
  • Dorado-Morales, Pedro|||0000-0001-5760-1999
  • Navarro, Susanna|||0000-0001-8160-9536
  • Ventura, Salvador|||0000-0002-9652-6351
  • Garnett, James A.
  • Lasa, Iñigo|||0000-0002-6625-9221
  • Valle, Jaione|||0000-0003-3115-0207
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.




Analysis of the association between polymorphisms in intergenic regions of Staphylococcus aureus genes involved in biofilm formation and periprosthetic joint infections

Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
  • Morales Laverde, Liliana Andrea
In this thesis, we have focused on studying variants found in IGRs adjacent to the most important genes involved in S. aureus biofilm formation; the icaADBCR locus, and the genes encoding the family of surface adhesins. For this purpose, we sequenced the whole genome of a collection of 71 S. aureus isolates from periprosthetic joint infections (PJI) and wound infections stored at the Clinical Bacteriological Laboratory of the Sahlgrenska University Hospital and at the Culture Collection University of Gothenburg (CCUG), respectively.
In the first chapter, we explored the regulatory regions of the icaADBCR locus to identify patterns that might be associated with an increased capacity of the isolates to produce PIA/PNAG and form a biofilm. This study compared the regulatory regions of the icaADBCR locus in the genomes of PJI and wound isolates with those in the genome of the reference strain MW2. From these analyses, strains were grouped based on the SNPs found in the IGRs of the operon and also within the coding region of the transcriptional regulator IcaR.
These regions showed high conservation rates, and no pattern associated with the origin of the isolates, either PJI or wounds, was detected. On the other hand, using transcriptional fusions between the regulatory region of the icaADBCR locus and the green fluorescent protein gene (gfp), we demonstrated that the expression of icaADBC genes was not affected by the presence of variations in IGRs. Notably, a SNP within the coding region of icaR, which results in an amino acid change in the transcriptional repressor IcaR V176E, led to a significant increase in the transcription of the icaADBC operon and the production of PIA/PNAG. Using a Galleria mellonella infection model, we were able to demonstrate a significant reduction in S. aureus virulence associated with the
increase in PIA/PNAG production.
In the second chapter, we focused on analyzing the association between SNPs in the promoter regions of genes encoding adhesion-related proteins with adhesins expression levels and therefore, the ability of the strain to adhere to medical devices. Genome analyses of PJI and wound isolates showed different profiles in the content of adhesin-encoding genes. Some of these, such as sasG and cna, were lineage-associated, and fifteen genes were present in the whole collection of strains. When the variability in the SNPs contained in regulatory regions that control the expression of each adhesin was investigated, different variation rates were found among the isolates. Following the same approach as in chapter I, based on transcriptional fusions between regulatory regions and the gfp gene, results showed that each genetic lineage contained a specific profile of adhesins expression under the same environmental condition.
Moreover, we developed a biomaterial-associated murine infection model together with a metagenomic analysis to simultaneously compare the capacity of different S. aureus isolates to colonize medical implants.
In summary, our results evidenced that SNPs in the IGRs flanking the genes encoding factors important for biofilm development may contribute to the generation of variability in the capacity of S. aureus to colonize medical implants.
In particular, our results revealed that IGRs controlling the expression of the icaADBC locus and production of the PIA/PNAG exopolysaccharide are highly conserved and that very few silent SNPs can be detected between strains. On the
contrary, SNPs in the IGRs of genes encoding surface adhesins provide a profile of proteins expression that is specific for each S. aureus clonal complex (CC).
Altogether, these studies emphasize the importance of investigating the potential impact of SNPs inside IGRs on gene expression and specific bacterial traits, such as pathogen colonization success., European Union's H2020 research and innovation programme under Marie Sklodowska-Curie grant agreement No 801586; Spanish Ministry of Economy, Industry and Competitiveness grant BIO2017-83035-R; Spanish Ministry of Science and Innovation grant PID2020-113494RB-I00., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)




σB Inhibits Poly-N-Acetylglucosamine Exopolysaccharide Synthesis and Biofilm Formation in Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Valle Turrillas, Jaione
  • Echeverz, Maite
  • Lasa, Íñigo
Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity., Work in the Laboratory of Microbial Pathogenesis is funded by the Spanish Ministry of Science, Innovation and Universities (grants SAF2015-74267-JIN and BIO2017-83035-R [Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union])., 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




Advances in bacterial transcriptome understanding: From overlapping transcription to the excludon concept

Digital.CSIC. Repositorio Institucional del CSIC
  • Toledo-Arana, Alejandro
  • Lasa, Íñigo
In the last decade, the implementation of high-throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon., This review is a tribute to part of the research legacy of P. Cossart's lab, to whom the authors have had the honour of belonging. This work was supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC Consolidator Grant Agreement No. 646869) to A.T‐A. and Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union (BIO2017‐83035‐R) to I.L.




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

Digital.CSIC. Repositorio Institucional del CSIC
  • Taglialegna, Agustina
  • Matilla-Cuenca, Leticia
  • Dorado-Morales, Pedro
  • Navarro, Susanna
  • Ventura, Salvador
  • Garnett, James A.
  • Lasa, Íñigo
  • Valle Turrillas, Jaione
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.




Elevated c-di-GMP levels promote biofilm formation and biodesulfurization capacity of Rhodococcus erythropolis

Digital.CSIC. Repositorio Institucional del CSIC
  • Dorado-Morales, Pedro
  • Martínez, Igor
  • Rivero-Buceta, Virginia
  • Díaz, Eduardo
  • Bähre, Heike
  • Lasa, Íñigo
  • Solano, Cristina
15 p.-4 fig.-2 tab., Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di‐GMP (c‐di‐GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities., Peer reviewed




Revisiting Bap Multidomain Protein: More Than Sticking Bacteria Together

Digital.CSIC. Repositorio Institucional del CSIC
  • Valle Turrillas, Jaione
  • Fang, Xianyang
  • Lasa, Íñigo
One of the major components of the staphylococcal biofilm is surface proteins that assemble as scaffold components of the biofilm matrix. Among the different surface proteins able to contribute to biofilm formation, this review is dedicated to the Biofilm Associated Protein (Bap). Bap is part of the accessory genome of Staphylococcus aureus but orthologs of Bap in other staphylococcal species belong to the core genome. When present, Bap promotes adhesion to abiotic surfaces and induces strong intercellular adhesion by self-assembling into amyloid like aggregates in response to the levels of calcium and the pH in the environment. During infection, Bap enhances the adhesion to epithelial cells where it binds directly to the host receptor Gp96 and inhibits the entry of the bacteria into the cells. To perform such diverse range of functions, Bap comprises several domains, and some of them include several motifs associated to distinct functions. Based on the knowledge accumulated with the Bap protein of S. aureus, this review aims to summarize the current knowledge of the structure and properties of each domain of Bap and their contribution to Bap functionality., Work in the laboratories of JV and IL is supported by grants RTI2018-096011-B-I00 and BIO2017-83035-R from Spanish Ministry of Science, Innovation and Universities. Work in the laboratory of XF is supported by grants from the National Natural Science Foundation of China (No. 31872712) and the Beijing Advanced Innovation Center for Structural Biology., Peer reviewed




A Systematic Evaluation of the Two-Component Systems Network Reveals That ArlRS Is a Key Regulator of Catheter Colonization by Staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Burgui, Saioa
  • Gil, Carmen
  • Solano Goñi, Cristina
  • Lasa, Íñigo
  • Valle Turrillas, Jaione
Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.




Fitness cost evolution of natural plasmids of staphylococcus aureus

Digital.CSIC. Repositorio Institucional del CSIC
  • Dorado-Morales, Pedro
  • Garcillán-Barcia, M. Pilar
  • Lasa, Iñigo
  • Solano, Cristina
© 2021 Dorado-Morales et al., Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S. P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.