RESUMO
Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.
Assuntos
Clostridium botulinum/isolamento & purificação , Indústria Alimentícia/métodos , Alimentos em Conserva , Microbiologia da Água , Animais , Técnicas Bacteriológicas/métodos , Toxinas Botulínicas/biossíntese , Botulismo/diagnóstico , Botulismo/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática/métodos , Programas de Rastreamento/métodos , Camundongos , Reação em Cadeia da Polimerase/métodos , Estados UnidosRESUMO
Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine, thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active dichain form of the toxin. Nicking of the single chain BoNT/E to the dichain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 degrees C the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction-induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.
Assuntos
Toxinas Botulínicas/metabolismo , Clostridium botulinum , Endopeptidases/metabolismo , Toxinas Botulínicas/química , Toxinas Botulínicas Tipo A/metabolismo , Dicroísmo Circular , Dissulfetos/química , Ativação Enzimática , Modelos Moleculares , Oxirredução , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Proteína 25 Associada a Sinaptossoma/metabolismo , TemperamentoRESUMO
Botulism is a deadly disease caused by ingestion of the preformed neurotoxin produced from the anaerobic spore-forming bacteria Clostridium botulinum. Botulinum neurotoxins are the most poisonous toxins known and have been a concern in the food industry for a long time. Therefore, rapid identification of botulinum neurotoxin using molecular and biochemical techniques is an essential component in the establishment of coordinated laboratory response systems and is the focus of current research and development. Because of the extreme toxicity of botulinum neurotoxin, some confirmatory testing with the mouse bioassay is still necessary, but rapid methods capable of screening large numbers of samples are also needed. This review is focused on the development of several detection methods for botulinum neurotoxins in foods.