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.

The Type III Secretion System (T3SS) of many human pathogens is used to secrete bacterial proteins into the host cells via a so-called needle structure and thereby infect them. Bacteria using T3SS include Shigella, which can cause bacterial dysentery, Yersinia, which is known to transmit the plague, and Salmonella, which is known to cause severe diarrhea. In this project, we took a closer look on the structures of the needle complex in the T3SS of Salmonella, which is directly involved in the infection process. By using biochemical and genetic methods, we initially unraveled the structures at the molecular level. In the following the use of state-of-the-art imaging methods provided a unique insight into the structure of the needle complex. In collaboration with the University of KU Leuven (Netherlands), we started with the biochemical characterization of the needle complexes and found a way to stabilize a subcomponent of the complex in such a way that it was possible to isolate it biochemically and use it for structural investigations for the first time. The second part of the project dealt with the structural analysis of single components of the needle complex. The applied method of electron cryotomography (CryoET) is an imaging technique that allows to generate a high-resolution three-dimensional view of complex macromolecules, in this case the needle complexes, from numerous single images. Previous work has shown that the size of the data set has a decisive influence on the level of resolution. To facilitate the work with these large data sets, we have developed a software to automate the work with those large tomographic records and thus increase the efficiency of data processing. Thus, within the project a machine learning algorithm was designed and extensively trained. It enables the recognition of needle complexes from Salmonella and their differentiation from other macromolecular complexes. The new software is suitable to identify needle complexes automatically in 3D cryo tomograms and to make them available for further reconstruction. The evaluation of this method showed no differences to manual methods of identifying needle complexes.