Objective
- To provide the molecular basis of toxin action for a scientific validation of in vitro toxicity assays of vacuolating toxin of Helicobacter pylori (vacA), of tetanus neurotoxin (TeNT) and of botulinum neurotoxins (BoNTs).
- To develop in vitro assays of the toxicity of vacA.
- To develop in vitro assays of the enzymatic activity and of the neuronal toxicity of TeNT and of BoNTs.
Toxicity of bacterial protein toxins is currently determined by using mice and other animals. There is a general consensus within Europe that whenever possible, experimentation and testing on animals should be replaced by in vitro or other methods. The achievement of these objectives will save many animals and, at the same time, will effectively reduce costs of vaccine and toxin production. The vacuolating toxin released by Helicobacter pylori interferes with membrane trafficking at the late endosomal stage in a rab7 dependent manner. Structural information on this toxin will be obtained by spectroscopic techniques and biocomputing methods and its crystallization will be attempted. The nature of its receptor(s) and the pathway of cell entry will be studied via biochemical techniques. Its intracellular activity will be studied by microinjection and transfection experiments as well as with in vitro endosome fusion assays. Identification of cellular proteins implicated in its action will be attempted via yeast two hybrids and other molecular cell biology approaches. Information obtained in this section of the project will be used to develop and validate reproducible in vitro assays of the activity of this toxin. The cellular effects of intoxication with tetanus and botulinum neurotoxins will be studied with various morphofunctional approaches as well as by immunofluorescence with selected antibodies. On this basis, cellular model systems to test the activity of these neurotoxins will be developed as well as novel assays of the metalloproteolytic activity of tetanus and botulinum neurotoxins.
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CSC - Cost-sharing contractsCoordinator
35121 Padova
Italy
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Participants (4)
53100 Siena
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01307 Weissig - Dresden
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78464 Konstanz
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1050 Bruxelles
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