Examinando por Autor "Krapp, Adriana R."

Mostrando 1 - 5 de 5
  • Miniatura
    ÍtemAcceso Abierto
    A long-chain flavodoxin protects pseudomonas aeruginosa from oxidative stress and host bacterial clearance
    (Public Library of Science (PLOS), 2014-02-13) Moyano, Alejandro José; Tobares, Romina Alín; Rizzi, Yanina S.; Krapp, Adriana R.; Mondotte, Juan Alberto; Bocco, José Luis; Saleh, Maria-Carla; Carrillo, Néstor; Smania, Andrea M.
    Long-chain flavodoxins, ubiquitous electron shuttles containing flavin mononucleotide (FMN) as prosthetic group, play an important protective role against reactive oxygen species (ROS) in various microorganisms. Pseudomonas aeruginosa is an opportunistic pathogen which frequently has to face ROS toxicity in the environment as well as within the host. We identified a single ORF, hereafter referred to as fldP (for flavodoxin from P. aeruginosa), displaying the highest similarity in length, sequence identity and predicted secondary structure with typical long-chain flavodoxins. The gene was cloned and expressed in Escherichia coli. The recombinant product (FldP) could bind FMN and exhibited flavodoxin activity in vitro. Expression of fldP in P. aeruginosa was induced by oxidative stress conditions through an OxyR-independent mechanism, and an fldP-null mutant accumulated higher intracellular ROS levels and exhibited decreased tolerance to H2O2 toxicity compared to wild-type siblings. The mutant phenotype could be complemented by expression of a cyanobacterial flavodoxin. Overexpression of FldP in a mutT-deficient P. aeruginosa strain decreased H2O2-induced cell death and the hypermutability caused by DNA oxidative damage. FldP contributed to the survival of P. aeruginosa within cultured mammalian macrophages and in infected Drosophila melanogaster, which led in turn to accelerated death of the flies. Interestingly, the fldP gene is present in some but not all P. aeruginosa strains, constituting a component of the P. aeruginosa accessory genome. It is located in a genomic island as part of a self-regulated polycistronic operon containing a suite of stress-associated genes. The collected results indicate that the fldP gene encodes a long-chain flavodoxin, which protects the cell from oxidative stress, thereby expanding the capabilities of P. aeruginosa to thrive in hostile environments.
  • Miniatura
    ÍtemAcceso Abierto
    Determinants of cofactor specificity for the glucose-6-phosphate dehydrogenase from Escherichia coli: simulation, kinetics and evolutionary studies
    (Public Library of Science (PLOS), 2016-03-24) Fuentealba, Matías; Muñoz, Rodrigo; Maturana, Pablo; Krapp, Adriana R.; Cabrera, Ricardo
    Glucose 6-Phosphate Dehydrogenases (G6PDHs) from different sources show varying specificities towards NAD+ and NADP+ as cofactors. However, it is not known to what extent structural determinants of cofactor preference are conserved in the G6PDH family. In this work, molecular simulations, kinetic characterization of site-directed mutants and phylogenetic analyses were used to study the structural basis for the strong preference towards NADP+ shown by the G6PDH from Escherichia coli. Molecular Dynamics trajectories of homology models showed a highly favorable binding energy for residues K18 and R50 when interacting with the 2'-phosphate of NADP+, but the same residues formed no observable interactions in the case of NAD+. Alanine mutants of both residues were kinetically characterized and analyzed with respect to the binding energy of the transition state, according to the kcat/KM value determined for each cofactor. Whereas both residues contribute to the binding energy of NADP+, only R50 makes a contribution (about -1 kcal/mol) to NAD+ binding. In the absence of both positive charges the enzyme was unable to discriminate NADP+ from NAD+. Although kinetic data is sparse, the observed distribution of cofactor preferences within the phylogenetic tree is sufficient to rule out the possibility that the known NADP+-specific G6PDHs form a monophyletic group. While the β1-α1 loop shows no strict conservation of K18, (rather, S and T seem to be more frequent), in the case of the β2-α2 loop, different degrees of conservation are observed for R50. Noteworthy is the fact that a K18T mutant is indistinguishable from K18A in terms of cofactor preference. We conclude that the structural determinants for the strict discrimination against NAD+ in the case of the NADP+-specific enzymes have evolved independently through different means during the evolution of the G6PDH family. We further suggest that other regions in the cofactor binding pocket, besides the β1-α1 and β2-α2 loops, play a role in determining cofactor preference.
  • Miniatura
    ÍtemEmbargo
    Diseño de un biosensor no invasivo para detectar el estado redox del NADP(H) en células vivas
    (2022) Molinari, Pamela Estefanía; Carrillo, Néstor; Krapp, Adriana R.
    Los procesos redox son esenciales para el desarrollo y la supervivencia de los seres vivos. Dentro de los diferentes pares redox, el NADP(H) cumple un rol central, proporcionando equivalentes reductores a múltiples vías biosintéticas, reguladoras y antioxidantes en todos los organismos vivos. No existe hasta ahora un procedimiento para monitorear selectivamente el estado redox de NADP(H) in vivo. La mayoría de los métodos existentes son invasivos o destructivos y/o se basan en ensayos in vitro. En este trabajo de tesis, fue diseñado y caracterizado un sensor no invasivo para determinar la fracción NADPH/NADP+ basado en las propiedades de roGFP2, una proteína verde fluorescente sensible a la reducción-oxidación, cuyas formas oxidada y reducida se excitan preferencialmente a dos longitudes de onda diferentes lo cual permite que sea ratiométrica y que se puedan realizar determinaciones basadas en la relación entre las dos excitaciones. El sensor roGFP2 fue fusionado a una proteína específica para NADP(H), la tiorredoxina reductasa C dependiente de NADPH (NTRC), que posee un dominio tiorredoxina C-terminal, y fue denominado NERNST (del inglés, NADP(H)-estimating ratiometric non destructive sensing tool). NERNST fue expresado en células de E. coli y caracterizado in vitro. Luego, fueron realizados ensayos in vivo a modo de pruebas de concepto en diferentes sistemas. Las propiedades de NERNST fueron investigadas mediante microscopía confocal en E. coli, pudiéndose estimar el potencial redox de NADP(H) en células que crecen en diferentes regímenes metabólicos. Además, fueron generadas plantas transgénicas estables de Arabidopsis y tabaco expresando el gen que codifica para NERNST, direccionando los productos a cloroplastos y a citosol, y fue analizada la respuesta del biosensor ante diferentes situaciones ambientales adversas, así como también en diferentes órganos de las plantas transformantes. Además, NERNST pudo ser expresado transitoriamente en plantas de N. benthamiana. Estudios en protoplastos aislados de hojas de plantas de Arabidopsis y tabaco transformadas de forma estable mostraron señales fluorescentes típicas de NERNST tanto en cloroplastos como en citosol, permitiendo estimaciones de ENADP(H) comparables a las obtenidas en hojas enteras. Resultados similares fueron obtenidos con protoplastos de plantas salvajes de Arabidopsis que fueron transformados transitoriamente usando los vectores empleados para el desarrollo de las anteriores. NERNST fue expresado en el citosol de células humanas en cultivo (HeLa y HEK-293T), y también fue direccionado a mitocondrias. El sensor fue empleado para estimar variaciones en el estado redox intracelular de NADP(H) por medio de la sobreexpresión de la NADPH oxidasa, que consume NADPH, y la inhibición de la glucosa-6-fosfato deshidrogenasa, que lo produce, así como la exposición a diferentes regímenes metabólicos. Por último, NERNST fue analizado en larvas de pez cebra como modelo de animal entero en conjunto con el sensor de H2O2, HyPerRed, pudiéndose detectar cambios en el pool de NADP(H) en respuesta a lesiones. En conjunto, los ensayos confirmaron que el biosensor NERNST refleja efectivamente los cambios esperados en el estado redox del pool de NADP(H) en todos los organismos ensayados.
  • Miniatura
    ÍtemAcceso Abierto
    NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems
    (Nature Research, 2023-06-06) Molinari, Pamela Estefanía; Krapp, Adriana R.; Weiner, Andrea María Julia; Beyer, Hannes M.; Kondadi, Arun Kumar; Blomeier, Tim; López, Melina; Bustos Sanmamed, Pilar; Tevere, Evelyn; Weber, Wilfried; Reichert, Andreas S.; Calcaterra, Nora B.; Beller, Mathias; Carrillo, Néstor; Zurbriggen, Matias D.
  • Miniatura
    ÍtemAcceso Abierto
    Reactive oxygen species generated in chloroplasts contribute to tobacco leaf infection by the necrotrophic fungus Botrytis cinerea
    (Society for Experimental Biology, 2017-10) Rossi, Franco R.; Krapp, Adriana R.; Bisaro, Fabiana; Maiale, Santiago J.; Pieckenstain, Fernando L.; Carrillo, Néstor
    Reactive oxygen species (ROS) play fundamental roles in plant responses to pathogen infection, including modulation of cell death processes and defense-related gene expression. Cell death triggered as part of the hypersensitive response enhances resistance to biotrophic pathogens, but favors the virulence of necrotrophs. Even though the involvement of ROS in the orchestration of defense responses is well established, the relative contribution of specific subcellular ROS sources to plant resistance against microorganisms with different pathogenesis strategies is not completely known. The aim of this work was to investigate the role of chloroplastic ROS in plant defense against a typical necrotrophic fungus, Botrytis cinerea. For this purpose, we used transgenic Nicotiana tabacum (tobacco) lines expressing a plastid-targeted cyanobacterial flavodoxin (pfld lines), which accumulate lower chloroplastic ROS in response to different stresses. Tissue damage and fungal growth were significantly reduced in infected leaves of pfld plants, as compared with infected wild-type (WT) counterparts. ROS build-up triggered by Botrytis infection and associated with chloroplasts was significantly decreased (70–80%) in pfld leaves relative to the wild type. Phytoalexin accumulation and expression of pathogenesis-related genes were induced to a lower degree in pfld plants than in WT siblings. The impact of fungal infection on photosynthetic activity was also lower in pfld leaves. The results indicate that chloroplast-generated ROS play a major role in lesion development during Botrytis infection. This work demonstrates that the modulation of chloroplastic ROS levels by the expression of a heterologous antioxidant protein can provide a significant degree of protection against a canonical necrotrophic fungus.