In vitro reconstitution of protein secretion through the Type 3 secretion system

The type III secretion system is a specialized protein export pathway. Its central component is the injectisome, a miniature syringe that sticks out of bacterial cells. Pathogenic bacteria such as Yersinia, Shigella, Pseudomonas, enteropathogenic/enterohemorrhagic E. coli (EPEC/EHEC) and Salmonella, the causative agent for many diseases known to animals or humans, use it to deliver toxins directly into eukaryotic cells. To control pathogenicity we need to understand what the injectisome looks like and how it works. For many years scientists have tried to do this in living bacteria by removing individual genes that encode injectisome components and by looking at it with powerful microscopes. We now propose a bold step to take these studies to the next level by setting up for the first time an in vitro reconstitution approach. This entails taking the individual components apart, away from the dizzying complexity of the cell and putting them back together again in a tube. Reconstitution is seminal in deciphering any biological processes. Many complementary approaches will be followed by the Flemish and Austrian labs that join forces in creating unique European synergies. They aim to learn which components take part in the inner workings of the injectisome, what is the order of events, how is metabolic energy used for the nanomachine to work and what does the machine look like in detail. Moreover, the project will make use of cryo electron microscopy, one of the most advanced and currently revolutionizing imaging techniques, to visualize the reconstituted injectiosme in action. By understanding the molecular mechanism of type III secretion system -mediated protein transport, we hope to provide a basis for the development of novel therapeutic strategies that will either inhibit its activity or modify the system for targeted drug delivery.