Structure-function analysis of Type IV secretion systems by cryo-electron microscopy

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Numerous diseases in humans, in other animals, and also in plants are caused by bacteria. The diseases are extremely diverse including food poisoning, tooth ache, anthrax, and even certain forms of cancer. Bacteria invented many various strategies to penetrate into different organisms to use their resources leading to illness of the host. These strategies, called bacterial pathogenicity, include transport of different important proteins and their complexes across the bacterial and cell membranes. To transfer toxic molecules and other proteins into target cells bacteria developed several types of special systems that are named as secretion systems. One of them that is characterised as a Type IV secretion (T4S) system is exceptionally adaptable to different conditions and able to transport large macromolecular complexes. For example, the human pathogen Helicobacter pylori, which plays a major role in the pathogenesis of peptic ulcers and gastric cancer, encodes a T4S system, the Cag-T4S system, which mediates the injection of the toxin CagA. T4S systems are macromolecular assemblies usually composed of 12 proteins (VirB1-11 and VirD4). Most T4S systems have three dedicated ATPases (VirD4 (or the coupling protein), VirB11, and VirB4) that play essential roles in supplying the energy for substrate translocation and apparatus assembly. Significant progress has been made in the past decade in our understanding of T4S systems: several structural elements were studied by crystallographic methods and the overall architecture of the T4S system core organisation was revealed by methods of electron microscopy. However, larger complexes remain to be investigated, notably a fully assembled T4S system containing all 12 components. Moreover, the dynamic nature of T4S systems remains to be revealed, including the exact role each ATPase plays during the secretion process. It has been shown that the ATPases VirD4, VirB11 and VirB4 interact with each other, and they are involved into substrate transfer process. However, very little is known concerning the details of the interactions of ATPases with each other and with the rest of the T4S apparatus. The overall aim of the project is to reveal the structural basis of interactions and signalling between ATPases and the central core. Combination of different methods such as molecular genetics, biochemistry, crystallography, and structural electron microscopy will reveal a total architecture of the T4S system and interactions between its components within this intricate system. Understanding how T4S system functions is a subject of this proposal and an important step for development of better treatment, and prevention of infectious diseases.