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Toledo Arana, Alejandro

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Toledo Arana

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Alejandro

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Instituto de Agrobiotecnología (IdAB)

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0000-0001-8148-6281

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5497

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Now showing 1 - 10 of 15
  • PublicationOpen Access
    Relevant role of fibronectin-binding proteins in Staphylococcus aureus biofilm-associated foreign-body infections
    (American Society for Microbiology, 2009) Vergara Irigaray, Marta; Valle Turrillas, Jaione; Merino Barberá, Nekane; Latasa Osta, Cristina; García Martínez, Begoña; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Toledo Arana, Alejandro; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant.
  • PublicationOpen Access
    Coordinated cyclic-di-GMP repression of salmonella motility through YcgR and cellulose
    (American Society for Microbiology, 2013) Zorraquino Salvo, Violeta; García Martínez, Begoña; Latasa Osta, Cristina; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: 1312/2010
    Cyclic di-GMP (c-di-GMP) is a secondary messenger that controls a variety of cellular processes, including the switch between a biofilm and a planktonic bacterial lifestyle. This nucleotide binds to cellular effectors in order to exert its regulatory functions. In Salmonella, two proteins, BcsA and YcgR, both of them containing a c-di-GMP binding PilZ domain, are the only known c-di-GMP receptors. BcsA, upon c-di-GMP binding, synthesizes cellulose, the main exopolysaccharide of the biofilm matrix. YcgR is dedicated to c-di-GMP-dependent inhibition of motility through its interaction with flagellar motor proteins. However, previous evidences indicate that in the absence of YcgR, there is still an additional element that mediates motility impairment under high c-di-GMP levels. Here we have uncovered that cellulose per se is the factor that further promotes inhibition of bacterial motility once high c-di-GMP contents drive the activation of a sessile lifestyle. Inactivation of different genes of the bcsABZC operon, mutation of the conserved residues in the RxxxR motif of the BcsA PilZ domain, or degradation of the cellulose produced by BcsA rescued the motility defect of ΔycgR strains in which high c-di-GMP levels were reached through the overexpression of diguanylate cyclases. High c-di-GMP levels provoked cellulose accumulation around cells that impeded flagellar rotation, probably by means of steric hindrance, without affecting flagellum gene expression, exportation, or assembly. Our results highlight the relevance of cellulose in Salmonella lifestyle switching as an architectural element that is both essential for biofilm development and required, in collaboration with YcgR, for complete motility inhibition.
  • PublicationOpen Access
    Evaluation of surface microtopography engineered by direct laser interference for bacterial anti-biofouling
    (2015) Valle Turrillas, Jaione; Burgui Erice, Saioa; Langheinrich, Denise; Gil Puig, Carmen; Solano Goñi, Cristina; Toledo Arana, Alejandro; Helbig, Ralf; Lasagni, Andrés; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1
    Biofilm formation by bacterial pathogens on the surface of medical and industrial settings is a 25 serious health problem. Modification of the biomaterial surface topography is a promising 26 strategy to prevent bacterial attachment and biofilm development. However, fabrication of 27 functional biomaterials at large scale with periodic network-topology is still problematic. In this 28 study, we use direct laser interference (DLIP), an easily scalable process, to modify polystyrene 29 surface (PS) topography at sub-micrometer scale. The resulting structure surfaces were 30 interrogated for their capacity to prevent adhesion and biofilm formation of the major human 31 pathogen Staphylococcus aureus. The results revealed that three-dimensional micrometer 32 periodic structures on PS have a profound impact on bacterial adhesion capacity. Thus, line- 33 and pillar-like topographical patterns enhanced S. aureus adhesion, whereas complex lamella 34 microtopography reduced S. aureus adhesion both in static and continuous flow culture 35 conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus 36 adhesion both when the surface is coated with human serum proteins in vitro and when the 37 material is implanted subcutaneously in a foreign-body associated infection model. Our results 38 establish that the DLIP technology can be used to functionalize polymeric surfaces for the 39 inhibition of bacterial adhesion to surfaces.
  • PublicationOpen Access
    The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus
    (Oxford University Press, 2018) Caballero Sánchez, Carlos; Menéndez Gil, Pilar; Catalán Moreno, Arancha; Vergara Irigaray, Marta; García Martínez, Begoña; Segura, Víctor; Irurzun Domínguez, Naiara; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
    RNA-binding proteins (RBPs) are essential to finetune gene expression. RBPs containing the coldshock 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 RBPimmunoprecipitation- 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 posttranscriptionally 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.
  • PublicationOpen Access
    Genome-wide antisense transcription drives mRNA processing in bacteria
    (National Academy of Sciences, 2011) Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; Dobin, Alexander; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Fagegaltier, Delphine; Penadés, José R.; Valle Turrillas, Jaione; Solano Goñi, Cristina; Gingeras, Thomas R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.
  • PublicationOpen Access
    The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation
    (American Society for Microbiology, 2001) Toledo Arana, Alejandro; Valle Turrillas, Jaione; Solano Goñi, Cristina; Arrizubieta Balerdi, María Jesús; Cucarella, Carme; Lamata, Marta; Amorena Zabalza, Beatriz; Leiva, José; Penadés, José R.; Lasa Uzcudun, Íñigo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.
  • PublicationOpen Access
    Biofilm switch and immune response determinants at early stages of infection
    (Elsevier (Cell Press), 2013) Valle Turrillas, Jaione; Solano Goñi, Cristina; García Martínez, Begoña; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1; Gobierno de Navarra / Nafarroako Gobernua: IIM13329.RI1; Gobierno de Navarra / Nafarroako Gobernua: 1312/2010
    Biofilm development is recognized as a major virulence factor underlying most chronic bacterial infections. When a biofilm community is established, planktonic cells growing in the surroundings of a tissue switch to a sessile lifestyle and start producing a biofilm matrix. The initial steps of in vivo biofilm development are poorly characterized and difficult to assess experimentally. A great amount of in vitro evidence has shown that accumulation of high levels of cyclic dinucleotides (c-di-NMPs) is the most prevalent hallmark governing the initiation of biofilm development by bacteria. As mentioned above, recent studies also link detection of c-di-NMPs by host cells with the activation of a type I interferon immune response against bacterial infections. We discuss here c-di-NMP signaling and the host immune response in the context of the initial steps of in vivo biofilm development.
  • PublicationOpen Access
    Protein A-mediated multicellular behavior in Staphylococcus aureus
    (American Society for Microbiology, 2008) Merino Barberá, Nekane; Toledo Arana, Alejandro; Vergara Irigaray, Marta; Valle Turrillas, Jaione; Solano Goñi, Cristina; Calvo, Enrique; Lopez, Juan Antonio; Foster, Timothy J.; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.
  • PublicationOpen Access
    Bap, a biofilm matrix protein of Staphylococcus aureus prevents cellular internalization through binding to GP96 host receptor
    (Public Library of Science, 2012) Valle Turrillas, Jaione; Latasa Osta, Cristina; Gil Puig, Carmen; Toledo Arana, Alejandro; Solano Goñi, Cristina; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilmassociated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.
  • PublicationOpen Access
    Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus
    (Public Library of Science, 2013) Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Villanueva San Martín, Maite; Bitarte Manzanal, Nerea; Saramago, Margarida; Domingues, Susana; Arraiano, Cecilia M.; Fechter, Pierre; Romby, Pascale; Valle Turrillas, Jaione; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    The presence of regulatory sequences in the 39 untranslated region (39-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 39-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 39-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 39-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 39-UTRs may play in controlling mRNA expression. We showed that base pairing between the 39- UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 39-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 39-UTR with the 59-UTR of the same mRNA.