Structural-Functional Characterization and Physiological Significance of Ferredoxin-NADP+ Reductase from Xanthomonas axonopodis pv. citri

Xanthomonas axonopodis pv. citri is a phytopathogen bacterium that causes severe citrus canker disease. Similar to other phytopathogens, after infection by this bacterium, plants trigger a defense mechanism that produces reactive oxygen species. Ferredoxin-NADP+ reductases (FNRs) are redox flavoenzymes that participate in several metabolic functions, including the response to reactive oxygen species. Xanthomonas axonopodis pv. citri has a gene (fpr) that encodes for a FNR (Xac-FNR) that belongs to the subclass I bacterial FNRs. The aim of this work was to search for the physiological role of this enzyme and to characterize its structural and functional properties. The functionality of Xac-FNR was tested by cross-complementation of a FNR knockout Escherichia coli strain, which exhibit high susceptibility to agents that produce an abnormal accumulation of •O2-. Xac-FNR was able to substitute for the FNR in E. coli in its antioxidant role. The expression of fpr in X. axonopodis pv. citri was assessed using semiquantitative RT-PCR and Western blot analysis. A 2.2-fold induction was observed in the presence of the superoxide-generating agents methyl viologen and 2,3-dimethoxy-1,4-naphthoquinone. Structural and functional studies showed that Xac-FNR displayed different functional features from other subclass I bacterial FNRs. Our analyses suggest that these differences may be due to the unusual carboxy-terminal region. We propose a further classification of subclass I bacterial FNRs, which is useful to determine the nature of their ferredoxin redox partners. Using sequence analysis, we identified a ferredoxin (XAC1762) as a potential substrate of Xac-FNR. The purified ferredoxin protein displayed the typical broad UV-visible spectrum of [4Fe-4S] clusters and was able to function as substrate of Xac-FNR in the cytochrome c reductase activity. Our results suggest that Xac-FNR is involved in the oxidative stress response of Xanthomonas axonopodis pv. citri and performs its biological function most likely through the interaction with ferredoxin XAC1762

Resistencia al tratamiento con sorafenib en el hepatocarcinoma celular: efectos de la inhibición de sirtuinas 1 y 2 en modelos in vitro

La resistencia a multidrogas (MDR) disminuye la eficacia de sorafenib, un importante tratamiento de primera línea para el carcinoma hepatocelular (HCC). Las sirtuinas (SIRTs) 1 y 2 están asociadas con la progresión tumoral y la MDR. En este trabajo se trataron cultivos 2D y 3D (que mimetizan las características de los tumores in vivo) de líneas celulares de HCC con sorafenib solo o en presencia de inhibidores de SIRTs 1 y 2 (cambinol o EX-527: tratamientos combinados). Los cultivos sometidos a los tratamientos combinados mostraron una mayor disminución de la proliferación (expresión de PCNA, ciclina D1 y Ki-67), migración e invasión celular, así como un aumento de la apoptosis (actividad de caspasas-3/7) en comparación con aquellos tratados solamente con sorafenib. Debido a que la desregulación del ciclo celular y el bloqueo de la apoptosis son mecanismos asociados a la MDR, la modulación encontrada en las proteínas PCNA, ciclina D1, Ki-67 y caspasas-3/7 por cambinol y EX-527 probablemente esté cumpliendo un rol en el aumento de la sensibilidad de las líneas celulares de HCC a sorafenib. Además, EX-527 redujo la expresión de MRP3 y BCRP en las células de HCC tratadas con sorafenib. Como los transportadores ABC contribuyen a la MDR, la modulación hallada podría estar influyendo también en mejorar la respuesta de las células de HCC a sorafenib. Cabe destacar que los tratamientos continuaron siendo efectivos al pasar de los cultivos 2D a los 3D, reforzando la relevancia clínica de este estudio. Los hallazgos presentados avalan una potencial aplicación de los inhibidores de SIRTs 1 y 2 en combinación con sorafenib, para reducir la resistencia de las células de HCC al fármaco.Fil: Delprato, Carla Beatriz D. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Área Morfología. Instituto de Fisiología Experimental (IFISE-CONICET); Argentina

KatG, the bifunctional catalase of xanthomonas citri subsp. citri, responds to hydrogen peroxide and contributes to epiphytic survival on citrus leaves

Xanthomonas citri subsp. citri (Xcc) is the bacterium responsible for citrus canker. This bacterium is exposed to reactive oxygen species (ROS) at different points during its life cycle, including those normally produced by aerobic respiration or upon exposition to ultraviolet (UV) radiation. Moreover, ROS are key components of the host immune response. Among enzymatic ROS-detoxifying mechanisms, catalases eliminate H2O2, avoiding the potential damage caused by this specie. Xcc genome includes four catalase genes. In this work, we studied the physiological role of KatG, the only bifunctional catalase of Xcc, through the construction and characterization of a modified strain (XcckatG), carrying an insertional mutation in the katG gene. First, we evaluated the involvement of KatG in the bacterial adaptive response to H2O2. XcckatG cultures exhibited lower catalase activity than those of the wild-type strain, and this activity was not induced upon treatment with sub-lethal doses of H2O2. Moreover, the KatG-deficient mutant exhibited decreased tolerance to H2O2 toxicity compared to wild-type cells and accumulated high intracellular levels of peroxides upon exposure to sub-lethal concentrations of H2O2. To further study the role of KatG in Xcc physiology, we evaluated bacterial survival upon exposure to UV-A or UV-B radiation. In both conditions, XcckatG showed a high mortality in comparison to Xcc wild-type. Finally, we studied the development of bacterial biofilms. While structured biofilms were observed for the Xcc wild-type, the development of these structures was impaired for XcckatG. Based on these results, we demonstrated that KatG is responsible for Xcc adaptive response to H2O2 and a key component of the bacterial response to oxidative stress. Moreover, this enzyme plays an important role during Xcc epiphytic survival, being essential for biofilm formation and UV resistance.Para citar este articulo: Tondo ML, Delprato ML, Kraiselburd I, Fernández Zenoff MV, Farías ME, Orellano EG (2016) KatG, the Bifunctional Catalase of Xanthomonas citri subsp. citri, Responds to Hydrogen Peroxide and Contributes to Epiphytic Survival on Citrus Leaves. PLoS ONE 11(3): e0151657. doi:10.1371/journal.pone.0151657Fil: Tondo, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Delprato, María Laura. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Kraiselburd, Ivana. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Fernández Zenoff, María Verónica. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Farías, María Eugenia. Planta Piloto de Procesos Industriales Microbiológicos (PROIMI -CONICET); Argentina.Fil: Orellano, Elena G. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina

Green Light to Plant Responses to Pathogens: The Role of Chloroplast Light-Dependent Signaling in Biotic Stress

Light has a key impact on the outcome of biotic stress responses in plants by providing most of the energy and many signals for the deployment of defensive barriers. Within this context, chloroplasts are not only the major source of energy in the light; they also host biosynthetic pathways for the production of stress hormones and secondary metabolites, as well as reactive oxygen species and other signals which modulate nuclear gene expression and plant resistance to pathogens. Environmental, and in particular, light-dependent regulation of immune responses may allow plants to anticipate and react more effectively to pathogen threats. As more information is gathered, increasingly complex models are developed to explain how light and reactive oxygen species signaling could interact with endogenous defense pathways to elicit efficient protective responses against invading microorganisms. The emerging picture places chloroplasts in a key position of an intricate regulatory network which involves several other cellular compartments. This article reviews current knowledge on the extent and the main features of chloroplast contribution to plant defensive strategies against biotic stress.Fil: Delprato, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Krapp, Adriana del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Carrillo, Nestor Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Effect of <i>katG</i> disruption on biofilm formation.

<p>(A) GFP-labeled Xcc strains were grown on chambered cover slides and visualized under confocal laser scanning microscopy (CLSM) after 2 days of bacterial growth. Left panels show the biofilms developed at the bottom of the chambered cover slides with a magnification of 400X and right panels show a 2X zoom of the regions marked in the previous panels. Scale bars, 50 μm. (B) Xcc strains were statically grown on glass tubes for 12 days at 28°C. Biofilm formation levels on the air-liquid interface were determined by crystal violet staining. The results show the means and standard deviations of a representative experiment with triplicate samples. The experiment was repeated three times with similar results in all cases.</p

Catalase activity of Xcc cultures in response to sub-lethal levels of hydrogen peroxide^a.

<p>Catalase activity of Xcc cultures in response to sub-lethal levels of hydrogen peroxide<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151657#t002fn001" target="_blank">^a</a>.</p

Catalase activity pattern in the Xcc<i>katG</i> mutant.

<p>Xcc wild-type (WT), Xcc<i>katG</i> (<i>katG</i>) and cXcc<i>katG</i> (<i>ckatG</i>) strains were grown aerobically in SB medium to early exponential phase (4 h), and soluble extracts were prepared as described in the experimental section. Equal amounts of protein (25 μg) were separated by 8% (w/v) non-denaturing PAGE and stained for catalase activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151657#pone.0151657.ref024" target="_blank">24</a>].</p

Sensitivity of Xcc<i>katG</i> to hydrogen peroxide.

<p>(A) Hydrogen peroxide resistance of pre-adapted Xcc cells. Exponential phase cultures of Xcc wild-type and <i>katG</i> mutant were adapted with the indicated concentrations of H₂O₂ for 60 min and then exposed to 1 mM H₂O₂ for 15 min. The number of CFU was determined for each culture before and after the treatment with 1 mM H₂O₂ by plating of appropriate dilutions. The percentage of survival was calculated as the number of CFU after treatment divided by the number of CFU prior to treatment ×100. Data represent mean ± standard deviation of three independent experiments. (B) ROS accumulation upon exposure to hydrogen peroxide. Bacteria were exposed to 100 μM H₂O₂ for 1 hour, and total peroxides (-OOH) were determined in cleared extracts using the FOX II assay as described in the experimental section. Measurements were carried out in triplicate for two independent experiments, and the results are expressed as means ± standard deviations. Statistical significant differences (P < 0.05, ANOVA) between wild-type and <i>katG</i> strains are indicated by an asterisk.</p

Pathogenicity and epiphytic fitness of Xcc<i>katG</i> in orange plants.

<p>(A) Growth of Xcc strains in the apoplastic space of orange leaves. Xcc WT, Xcc<i>katG</i> and cXcc<i>katG</i> cells were inoculated at 10⁵ CFU/mL in 10 mM MgCl₂ into the intercellular spaces of fully expanded orange leaves. Bacterial populations in leaf tissues were determined by serial dilution and plating. A representative leaf 20 days after inoculation with the three strains is shown in the lower inset. Left panel, adaxial side; right panel, abaxial side. Dashed lines indicate the infiltrated area. (B) Epiphytic populations of Xcc strains on orange leaves. Bacterial cells were released from the leaf surface by sonication followed by dilution plating. Experiments were performed in triplicate; values are expressed as means ± standard deviations. Statistical significant differences (P < 0.05, ANOVA) between wild-type and <i>katG</i> strains are indicated by an asterisk.</p