CONOTOX

Functional characterization of neuroactive toxins using an engineered bacterial type-III secretion system

 Partecipanti

Mappa

 Word cloud

Esplora la "nuvola delle parole (Word Cloud) per avere un'idea di massima del progetto.

 Obiettivo del progetto (Objective)

'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.'

Introduzione (Teaser)

Neurological toxins occur throughout the natural world and vary greatly in their effect on humans. Researchers have developed a high-throughput microbiological system to produce various neurotoxins and test their effects on the nervous system.

Descrizione progetto (Article)

Marine cone snails, genus Conus, carry venom that consists of over 100 different neurotoxins per species. These neurotoxins also have an array of effects on their prey. A system that allows for easy production and purification of these toxins will help scientists to study their targets, most often the ion channels in nerve cells.

The EU-funded 'Functional characterization of neuroactive toxins using an engineered bacterial type-III secretion system' (CONOTOX) project created such a rapid neurotoxin production and purification system. Researchers are engineering a bacterium, Salmonella enterica, to secrete neurotoxins into their growing environment, where they can be easily purified and tested.

To achieve this, the group investigated ways to improve secretion in S. enterica. Using these results, they created a modified S. enterica strain with improved protein secretion (neurotoxins are peptides, or small proteins). This approach was tested with a model neurotoxin (tetanus toxin) and was shown to be an effective system for neurotoxin production.

Researchers will now use this system to produce Conus neurotoxins. They will create a DNA library of neurotoxin molecules, produce and purify the neurotoxin peptides, and screen them for their effect on ion channels in nerve cells.

Ion channel function is a crucial but poorly understood part of the vertebrate nervous system. CONOTOX will produce a large variety of neurotoxins that can be used to improve our understanding of ion channels and how they function.