Functional characterization of neuroactive toxins .. (ConoTox)
Functional characterization of neuroactive toxins using an engineered bacterial type-III secretion system
Start date: Jul 1, 2012,
End date: Jun 30, 2014
Toxins occur in a broad diversity in nature and are produced by a wide array of organisms, including spiders, snails, or plants. Conotoxins are a class of toxins that are synthesized in the venom duct of the marine cone snail, genus Conus. The venom of each Conus species is composed of 100 different toxins that rapidly affect the prey organism at multiple targets. The majority of toxins target ligand-gated or voltage-gated ion channels. Each neuroactive peptide of the venom is highly specific for an individual channel isoform.The applicant proposes to develop in an interdisciplinary approach engineered bacteria that export neuroactive peptides and proteins into the surrounding media, readily available for subsequent characterization. The applicant will utilize the powerful flagellar type-III secretion system of Salmonella enterica for the expression and secretion of recombinant neuroactive peptides. The flagellar-specific type-III secretion apparatus selectively exports substrate proteins at a rate up to 10,000 amino acid residues per second and can be readily manipulated by genetic methods. The applicant will create a Salmonella peptide library based on cDNA isolated from the venom duct of cone snails and use the type-III secretion system as an export shuttle to transport non-flagellar peptides into the surrounding media. The media supernatant or (partially) purified peptides will be analyzed for neurotoxic activity by imaging cellular calcium levels of dorsal root ganglia cells.This innovative approach for the rapid purification of neuroactive compounds using secretion via the well-characterized flagellar type-III secretion apparatus will allow to identify and characterize novel toxins with distinct target specificity. Due to their unique mode of action and their ability to discriminate between different members of a large ion channel family, these toxins have an immense potential for neurobiological research and novel diagnostic or therapeutic applications.
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