Towards the application of Tc toxins as a universal protein translocation system

Tc toxins are large bacterial protein complexes that inject cytotoxic enzymes into target cells using a sophisticated syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigated whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely human Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, human Ras-binding domain of CRAF kinase, and tobacco etch virus protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to successfully take advantage of this new universal protein translocation system.