RESUMO
The tellurium oxyanion tellurite is toxic to most organisms because of its ability to generate oxidative stress. However, the detailed mechanism(s) how this toxicant interferes with cellular processes have yet to be fully understood. As part of our effort to decipher the molecular interactions of tellurite with living systems, we have evaluated the global metabolism of α-ketoglutarate a known antioxidant in Escherichia coli. Tellurite-exposed cells displayed reduced activity of the KG dehydrogenase complex (KGDHc), resulting in increased intracellular KG content. This complex's reduced activity seems to be due to decreased transcription in the stressed cells of sucA, a gene that encodes the E1 component of KGDHc. Furthermore, it was demonstrated that the increase in total reactive oxygen species and superoxide observed upon tellurite exposure was more evident in wild type cells than in E. coli with impaired KGDHc activity. These results indicate that KG may be playing a pivotal role in combating tellurite-mediated oxidative damage.
Assuntos
Escherichia coli/efeitos dos fármacos , Ácidos Cetoglutáricos/metabolismo , Estresse Oxidativo , Telúrio/toxicidade , Di-Hidrolipoamida Desidrogenase/genética , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Complexo Cetoglutarato Desidrogenase/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly.
Assuntos
Infecções por Birnaviridae/veterinária , Doenças dos Peixes/virologia , Vírus da Necrose Pancreática Infecciosa/genética , Biossíntese de Proteínas , RNA Viral/genética , Regiões 5' não Traduzidas , Animais , Infecções por Birnaviridae/virologia , Regulação Viral da Expressão Gênica , Vírus da Necrose Pancreática Infecciosa/química , Vírus da Necrose Pancreática Infecciosa/metabolismo , Sítios Internos de Entrada Ribossomal , Conformação de Ácido Nucleico , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , RNA Viral/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Salmo salar , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Tellurite is toxic to most microorganisms because of its ability to generate oxidative stress. However, the way in which tellurite interferes with cellular processes is not fully understood to date. In this line, it was previously shown that tellurite-exposed cells displayed reduced activity of the α-ketoglutarate dehydrogenase complex (α-KGDH), which resulted in α-ketoglutarate (α-KG) accumulation. In this work, we assessed if α-KG accumulation in tellurite-exposed E. coli could also result from increased isocitrate dehydrogenase (ICDH) and glutamate dehydrogenase (GDH) activities, both enzymes involved in α-KG synthesis. Unexpectedly both activities were found to decrease in the presence of the toxicant, an observation that seems to result from the decreased transcription of icdA and gdhA genes (encoding ICDH and GDH, resp.). Accordingly, isocitrate levels were found to increase in tellurite-exposed E. coli. In the presence of the toxicant, cells lacking icdA or gdhA exhibited decreased reactive oxygen species (ROS) levels and higher tellurite sensitivity as compared to the wild type strain. Finally, a novel branch activity of ICDH as tellurite reductase is presented.