Miguel E. Castro

A double-stranded RNA mycovirus confers hypovirulence-associated traits to Botrytis cinerea.

Botrytis cinerea CCg425 contains a 33-nm isometric mycovirus whose genome is a 6.8-kb double-stranded RNA (dsRNA) molecule. Virulence bioassays, performed by direct plug mycelial inoculation on bean plant leaves, showed that B. cinerea CCg425 displays less fungal aggressivity than B. cinerea CKg54, a virulent fungal strain that is not infected by dsRNA mycoviruses. B. cinerea CCg425 also showed lower laccase activity and conidiation rate than B. cinerea CKg54. Furthermore, infection of B. cinerea CKg54 with viral particles purified from B. cinerea CCg425 resulted in diminished virulence of the infected fungus. Collectively, our results indicate that mycovirus infection confers hypovirulence to the fungal host.

The dihydrolipoamide dehydrogenase of Aeromonas caviae ST exhibits NADH-dependent tellurite reductase activity.

Potassium tellurite (K(2)TeO(3)) is extremely toxic for most forms of life and only a limited number of organisms are naturally resistant to the toxic effects of this compound. Crude extracts prepared from the environmental isolate Aeromonas caviae ST catalize the in vitro reduction of TeO32- in a NADH-dependent reaction. Upon fractionation by ionic exchange column chromatography three major polypeptides identified as the E1, E2, and E3 components of the pyruvate dehydrogenase (PDH) complex were identified in fractions exhibiting tellurite-reducing activity. Tellurite reductase and pyruvate dehydrogenase activities co-eluted from a Sephadex gel filtration column. To determine which component(s) of the PDH complex has tellurite reductase activity, the A. caviae ST structural genes encoding for E1 (aceE), E2 (aceF), and E3 (lpdA) were independently cloned and expressed in Escherichia coli and their gene products purified. Results indicated that tellurite reductase activity lies almost exclusively in the E3 component, dihydrolipoamide dehydrogenase. The E3 component of the PDH complex from E. coli, Zymomonas mobilis, Streptococcus pneumoniae, and Geobacillus stearothermophilus also showed NADH-dependent tellurite reductase in vitro suggesting that this enzymatic activity is widely distributed among microorganisms.

Cysteine Metabolism-Related Genes and Bacterial Resistance to Potassium Tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.

Expression of Aeromonas caviae ST pyruvate dehydrogenase complex components mediate tellurite resistance in Escherichia coli.

Potassium tellurite (K(2)TeO(3)) is harmful to most organisms and specific mechanisms explaining its toxicity are not well known to date. We previously reported that the lpdA gene product of the tellurite-resistant environmental isolate Aeromonas caviae ST is involved in the reduction of tellurite to elemental tellurium. In this work, we show that expression of A. caviae ST aceE, aceF, and lpdA genes, encoding pyruvate dehydrogenase, dihydrolipoamide transacetylase, and dihydrolipoamide dehydrogenase, respectively, results in tellurite resistance and decreased levels of tellurite-induced superoxide in Escherichia coli. In addition to oxidative damage resulting from tellurite exposure, a metabolic disorder would be simultaneously established in which the pyruvate dehydrogenase complex would represent an intracellular tellurite target. These results allow us to widen our vision regarding the molecular mechanisms involved in bacterial tellurite resistance by correlating tellurite toxicity and key enzymes of aerobic metabolism.

Rapid isolation of mycoviral double-stranded RNA from Botrytis cinerea and Saccharomyces cerevisiae.

BackgroundIn most of the infected fungi, the mycoviruses are latent or cryptic, the infected fungus does not show disease symptoms, and it is phenotypically identical to a non-infected strain of the same species. Because of these properties, the initial stage in the search for fungi infected with mycoviruses is the detection of their viral genome, which in most of the described cases corresponds to double-stranded RNA (dsRNA). So to analyze a large number of fungal isolates it is necessary to have a simple and rapid method to detect dsRNA.ResultsA rapid method to isolate dsRNA from a virus-infected filamentous fungus, Botrytis cinerea, and from a killer strain of Saccharomyces cerevisiae using commercial minicolumns packed with CF11 cellulose was developed. In addition to being a rapid method, it allows to use small quantities of yeasts or mycelium as starting material, being obtained sufficient dsRNA quantity that can later be analyzed by agarose gel electrophoresis, treated with enzymes for its partial characterization, amplified by RT-PCR and cloned in appropriate vectors for further sequencing.ConclusionsThe method yields high quality dsRNA, free from DNA and ssRNA. The use of nucleases to degrade the DNA or the ssRNA is not required, and it can be used to isolate dsRNA from any type of fungi or any biological sample that contains dsRNA.

Expression of the yggE gene protects Escherichia coli from potassium tellurite-generated oxidative stress

Potassium tellurite is highly toxic to most forms of life and specific bacterial tellurite defense mechanisms are not fully understood to date. Recent evidence suggests that tellurite would exert its toxic effects, at least in part, through the generation of superoxide anion that occurs concomitantly with intracellular tellurite (Te4+) reduction to elemental tellurium (Teo). In this work the putative antioxidant role of YggE from Escherichia coli, a highly conserved protein in several bacterial species and whose function is still a matter of speculation, was studied. When exposed to tellurite, E. coli lacking yggE exhibited increased activity of superoxide dismutase and fumarase C, augmented levels of reactive oxygen species and high concentration of carbonyl groups in proteins. Upon genetic complementation with the homologous yggE gene these values were restored to those observed in the parental, isogenic, wild type strain, suggesting a direct participation of YggE in E. coli tolerance to tellurite.

Inhibitory Effect of Bridged Nucleosides on Thermus aquaticus DNA Polymerase and Insight into the Binding Interactions.

Modified nucleosides have the potential to inhibit DNA polymerases for the treatment of viral infections and cancer. With the hope of developing potent drug candidates by the modification of the 2’,4’-position of the ribose with the inclusion of a bridge, efforts were focused on the inhibition of Taq DNA polymerase using quantitative real time PCR, and the results revealed the significant inhibitory effects of 2’,4’-bridged thymidine nucleoside on the polymerase. Study on the mode of inhibition revealed the competitive mechanism with which the 2’,4’-bridged thymidine operates. With a Ki value of 9.7 ± 1.1 μM, the 2’,4’-bridged thymidine proved to be a very promising inhibitor. Additionally, docking analysis showed that all the nucleosides including 2’,4’-bridged thymidine were able to dock in the active site, indicating that the substrate analogs reflect a structural complementarity to the enzyme active site. The analysis also provided evidence that Asp610 was a key binding site for 2’,4’-bridged thymidine. Molecular dynamics (MD) simulations were performed to further understand the conformational variations of the binding. The root-mean-square deviation (RMSD) values for the peptide backbone of the enzyme and the nitrogenous base of the inhibitor stabilized within 0.8 and 0.2 ns, respectively. Furthermore, the MD analysis indicates substantial conformational change in the ligand (inhibitor) as the nitrogenous base rotated anticlockwise with respect to the sugar moiety, complemented by the formation of several new hydrogen bonds where Arg587 served as a pivot axis for binding formation. In conclusion, the active site inhibition of Taq DNA polymerase by 2’,4’-bridged thymidine suggests the potential of bridged nucleosides as drug candidates.

Abstract 411: Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA BRAF is a human gene encoding a serine/threonine protein kinase in the RAS-RAF-MEK-ERK signal pathway, which is important in regulating cellular responses to extracellular signals. Frequent mutations on BRAF gene have been detected in various types of human cancer, including malignant melanoma, non-small cell lung cancer, colorectal cancer, papillary carcinoma of the thyroid, ovarian cancer and hairy cell leukemia. Among all, a single point 1799T>A (V600E) transversion, the most common mutation on BRAF, results in increased kinase activity. It serves as a driver mutation in several types of cancer and confers increased sensitivity to tyrosine kinase inhibitors. Thus, detecting the BRAF V600E mutation may offer valuable information for cancer diagnosis, treatment selection, and prognosis. Here we report a rapid, highly sensitive method for the detection of BRAF V600 mutation using a bridged nucleic acid (BNA) clamp technology associated with quantitative PCR (qPCR). Due to the feature of enhanced affinity for hybridization template, BNA clamp is able to selectively block PCR reactions on the perfect match DNA sequence, while leaves the mismatch template unaffected. In this study several BNA clamps that bind to BRAF V600 wild-type (WT) template was designed, synthesized, and characterized. As compared to DNA clamp counterparts, these BNA clamps increased Tm values for the BRAF V600 WT by greater than 30°C. In addition, BNA clamps efficiently distinguished BRAF WT (perfect match) from V600E (mismatch) templates (deltaTm >12°C). In qPCR assays BNA clamps could suppress amplification of WT by >1,000-fold, while had little or no effects on the V600E mutant. Such BNA clamps allowed us to detect 1.0% or lower levels for the BRAF V600E mutant in a wild-type background. These results indicate that BNA clamp-based qPCR technology offers a new tool for rapid, sensitive detection of BRAF V600E mutation. The technology may be adapted for detection of mutations of a variety of biomarker genes. Citation Format: Xiaoyun Liu, Leticia Loredo, Houquan Dai, Aaron Castro, Yuewei Zhao, Sung Kun Kim, Austin Dinkel, Miguel Castro. Sensitive detection of BRAF V600E mutation by bridged nucleic acid (BNA)-mediate real-time PCR assay. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 411.