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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 - 6 of 6
  • 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
    Staphylococcus aureus develops an alternative, ica-independent biofilm in the absence of the arlRS two-component system
    (American Society for Microbiology, 2005) Toledo Arana, Alejandro; Merino Barberá, Nekane; Vergara Irigaray, Marta; Débarbouillé, Michel; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    The biofilm formation capacity of Staphylococcus aureus clinical isolates is considered an important virulence factor for the establishment of chronic infections. Environmental conditions affect the biofilm formation capacity of S. aureus, indicating the existence of positive and negative regulators of the process. The majority of the screening procedures for identifying genes involved in biofilm development have been focused on genes whose presence is essential for the process. In this report, we have used random transposon mutagenesis and systematic disruption of all S. aureus two-component systems to identify negative regulators of S. aureus biofilm development in a chemically defined medium (Hussain-Hastings-White modified medium [HHWm]). The results of both approaches coincided in that they identified arlRS as a repressor of biofilm development under both steady-state and flow conditions. The arlRS mutant exhibited an increased initial attachment as well as increased accumulation of poly-N-acetylglucosamine (PNAG). However, the biofilm formation of the arlRS mutant was not affected when the icaADBC operon was deleted, indicating that PNAG is not an essential compound of the biofilm matrix produced in HHWm. Disruption of the major autolysin gene, atl, did not produce any effect on the biofilm phenotype of an arlRS mutant. Epistatic experiments with global regulators involved in staphylococcal-biofilm formation indicated that sarA deletion abolished, whereas agr deletion reinforced, the biofilm development promoted by the arlRS mutation.
  • 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
    Calcium inhibits bap-dependent multicellular behavior in Staphylococcus aureus
    (American Society for Microbiology, 2004) Arrizubieta Balerdi, María Jesús; Toledo Arana, Alejandro; Amorena Zabalza, Beatriz; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
    Bap (biofilm-associated protein) is a 254-kDa staphylococcal surface protein implicated in formation of biofilms by staphylococci isolated from chronic mastitis infections. The presence of potential EF-hand motifs in the amino acid sequence of Bap prompted us to investigate the effect of calcium on the multicellular behavior of Bap-expressing staphylococci. We found that addition of millimolar amounts of calcium to the growth media inhibited intercellular adhesion of and biofilm formation by Bap-positive strain V329. Addition of manganese, but not addition of magnesium, also inhibited biofilm formation, whereas bacterial aggregation in liquid media was greatly enhanced by metal-chelating agents. In contrast, calcium or chelating agents had virtually no effect on the aggregation of Bap-deficient strain M556. The biofilm elicited by insertion of bap into the chromosome of a biofilm-negative strain exhibited a similar dependence on the calcium concentration, indicating that the observed calcium inhibition was an inherent property of the Bap-mediated biofilms. Site-directed mutagenesis of two of the putative EF-hand domains resulted in a mutant strain that was capable of forming a biofilm but whose biofilm was not inhibited by calcium. Our results indicate that Bap binds Ca2+ with low affinity and that Ca2+ binding renders the protein noncompetent for biofilm formation and for intercellular adhesion. The fact that calcium inhibition of Bap-mediated multicellular behavior takes place in vitro at concentrations similar to those found in milk serum supports the possibility that this inhibition is relevant to the pathogenesis and/or epidemiology of the bacteria in the mastitis process.
  • PublicationOpen Access
    Genetic reductionist approach for dissecting individual roles of GGDEF proteins within the c-di-GMP signaling network in Salmonella
    (National Academy of Sciences, 2009) Solano Goñi, Cristina; García Martínez, Begoña; Latasa Osta, Cristina; Toledo Arana, Alejandro; Zorraquino Salvo, Violeta; Valle Turrillas, Jaione; Casals, Joan; Pedroso, Enrique; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
    Bacteria have developed an exclusive signal transduction system involving multiple diguanylate cyclase and phosphodiesterase domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) that modulate the levels of the same diffusible molecule, 3 -5 -cyclic diguanylic acid (c-di-GMP), to transmit signals and obtain specific cellular responses. Current knowledge about c-di- GMP signaling has been inferred mainly from the analysis of recombinant bacteria that either lack or overproduce individual members of the pathway, without addressing potential compensatory effects or interferences between them. Here, we dissected c-di-GMP signaling by constructing a Salmonella strain lacking all GGDEF-domain proteins and then producing derivatives, each restoring 1 protein. Our analysis showed that most GGDEF proteins are constitutively expressed and that their expression levels are not interdependent. Complete deletion of genes encoding GGDEFdomain proteins abrogated virulence, motility, long-term survival, and cellulose and fimbriae synthesis. Separate restoration revealed that 4 proteins from Salmonella and 1 from Yersinia pestis exclusively restored cellulose synthesis in a c-di-GMP–dependent manner, indicating that c-di-GMP produced by different GGDEF proteins can activate the same target. However, the restored strain containing the STM4551-encoding gene recovered all other phenotypes by means of gene expression modulation independently of c-di-GMP. Specifically, fimbriae synthesis and virulence were recovered through regulation of csgD and the plasmid-encoded spvAB mRNA levels, respectively. This study provides evidence that the regulation of the GGDEF-domain proteins network occurs at 2 levels: a level that strictly requires c-di-GMP to control enzymatic activities directly, restricted to cellulose synthesis in our experimental conditions, and another that involves gene regulation for which c-di-GMP synthesis can be dispensable.
  • 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.