Cellular uptake of the Rho-activating toxins CNF1 and CNFY and of further deamidases

• Applicant: Professorin Dr. Gudula Schmidt
• Subject Area: Pharmacology ; Cell Biology
• Term: since 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 261620687

For many years my group studied bacterial toxins (deamidases), which constitutively activate Rho GTPases or the Gα-subunit of heterotrimeric G-proteins, respectively. We analyzed the molecular mechanisms of 5 members of the CNF family (Cytotoxic Necrotizing Factors 1, 2, 3, CNFY and CNFS) produced by Escherichia coli, Yersinia pseudotuberculosis (CNFY) and Salmonella enterica (CNFS), respectively. Moreover, we studied Pasteurella multocida toxin (PMT) and Bordetella Dermo-Necrotzing toxin (DNT). Besides the molecular mechanism, the knowledge about cell specificity (cellular receptor) and uptake route into mammalian cells is essential for the cell-biological use and pharmacological inhibition of the powerful molecular tools.For the identification of cellular protein receptors, biochemical methods have been used to purify high affinity membrane proteins. Indeed, some receptors like the CNF1 binding Lu/BCAM (Lutheran/ Basal Cell Adhesion Molecule) have been identified. Moreover, we characterized proteoglycans as landing platform for the toxins. For more effective and straight forward strategy for receptor identification, we established a new method based on a lentiviral knockout library. Therefore, we generated two stable Cas9 expressing mouse cell lines and a protocol in which allows knockout of statistically one gene per cell. Using a chimeric toxin PMT-DTA (catalytic domain of diphtheria toxin), we already performed the first screens and identified the following genes which seem to be necessary for binding, uptake and action of the chimeric toxin: two genes for diphthamide synthesis (target amino acid for DTA, showing functionality of out assay), LDL Receptor Related Protein 1 (LRP1) and a subunit of the Oligosaccharyltransferase Complex (Ostc). In first experiments, LRP1-knockout cells showed resistance towards PMT-DTA. We plan to study the relevance of LRP-1 and Ostc for binding and uptake of PMT and to define the exact interacting amino acids. Moreover, we want to use our genetic screen for identification of further toxin-receptor interaction partners, especially for Yersinia CNFY.

• DFG Programme: Research Grants