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

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.

Pathogenicity of bacteria is closely linked to secretion machineries which provide and secure both transportation and injection of toxic molecules into target cells. Among several secretion systems existing in bacteria, the type IV secretion (T4S) systems are exceptionally adaptable to different conditions in bacteria. All known T4S systems are evolutionary related: they have similar major components and share a common requirement for proteins that utilize ATP as an energy source to drive transport of macromolecules. T4S systems are huge dynamic macromolecular complexes that comprise 12 proteins VirB1-11, and VirD4. The assembly is powered by three ATPases: VirD4, VirB11 and VirB4, which interact with each other. It has been shown that the ATPases promote substrate transfer; however, very little is known concerning the details of their interactions with a substrate, and with the core of T4S systems. In this study we aim to reveal structures of the T4S system core complexed with individual ATPases VirB4, VirD4, and VirB11, and their complexes with additional components VirB3 and VirB8. That will help us to understand principles of interactions between the components and to trace the signalling pathway within the T4S system.
To reveal a structural basis of the T4SS function we will combine methods of structural cryo electron microscopy, crystallography, NMR, and bioinformatics. All available atomic structures (VirB11 ATPase, VirD4 ATPase, VirB8 and VirB10 periplasmic domains, and the OM complex) will be docked into the electron density maps to identify locations of the protein components and their conformational changes upon binding. T4S system activity will be monitored by specially designed T4S system assay based on conjugation. The essential feature of our research programme is that cell microbiological approaches that form the basis of T4S infectivity studies are complementing our structural biology work to address crucial questions in the infection process.

T4S systems are unique among the known macromolecular translocation systems, now numbering at least seven, in this broad phylogenetic distribution. How these machines initiate the formation of and facilitate translocation of DNA and proteins across intercellular junctions is a crucial question, especially in view of increasing levels of antibiotic resistance of bacteria. The T4S systems are key therapeutic targets; this work could impact on drug development by the pharmaceutical industry. This remains a priority area since the T4S system inhibitors would have the added advantage of inhibiting propagation of antibiotic resistance genes and therefore the spread of hospital-acquired infections.
The immediate beneficiaries include researchers in academia (national and international) and in the private commercial sector (pharmaceutical companies). Interested academics are:
(i) Those in the immediate research area of bacterial T4S systems that form supramolecular protein complexes that are responsible for transporting DNA or protein substrates across the bacterial cell envelope and, in many cases, also across eukaryotic target cell membranes.
(ii) Those in the general research areas of pathogenic bacteria.
(iii) Those in the research areas of different T4S systems that are responsible for horizontal gene transfer, and the cytotoxin-associated gene (Cag) T4S system for interactions with various host cells.
(iv) Those who seek methodological advances in single particle analysis by electron microscopy.
(v) Structural biologists employing diverse biophysical techniques to relate structure to function.
Long-term direct beneficiaries would include:
(i) Bacteriologists, who are deeply interested in revelation how these machines (T4S systems) induce the formation of and mediate translocation across intercellular junctions, especially in view of the striking diversity of prokaryotic cell envelopes.
(ii) Medical scientists.
(iii) The wider population who will benefit from improved health and wealth that would accompany the understanding and decreasing the bacterial antibiotic resistance.
The work will advance our understanding the mechanisms of transport of effector proteins and DNAs into host cells. T4S systems are multi-compound assemblies. While some components and their complexes have been visualized (by us) by crystallography and electron microscopy, the structural and molecular basis of substrate recruitment and transport during cell conjugation is yet to be established. Although, there is already significant structural and functional data for T4S systems that could facilitate a rational approach to drug design, this structure-function study is crucial for dissecting and understanding these supramolecular assemblies for therapeutic targets. The prospect that this and any new data emerging from our proposed study can be applied immediately is realistic. The nation's health and wealth would improve significantly if the means of inhibiting propagation of antibiotic resistance will be revealed and targets identified. It will lead to the alleviation of the disease burden in animals and humans. Selective anti-bacterial drugs would have a direct impact on national health and reduce the financial burden on public health resources.
Pharmaceutical companies that develop anti-bacterial drugs would derive wealth directly from their commercial products. There is also the potential for patentable results as assays for screening therapeutic agents. There will also be benefits from the continued training of postdoctoral research fellows and the development of their profession skills and creativity that could be integrated into any commercial or academic enterprise requiring a highly skilled structural biologist or protein biochemist. Many of the skills that will develop, such as time management, team working, communication and technical, are also transferable between employment sectors.