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

Lead Research Organisation: Birkbeck College
Department Name: Biological Sciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description practical courses on electron microscopy
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
Impact Providing teaching materials,, raising awareness of importance of safety in research, importance of biological sciences for medicine, public services and environmental sustainability
 
Description Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM): Supporting the software infrastructure for cryoEM techniques. 2015- 2020.
Amount £1,177,000 (GBP)
Funding ID MR/N009614/1 
Organisation Medical Research Council (MRC) 
Department MRC Partnership Grant
Sector Public
Country United Kingdom
Start  
 
Description State of the art cryo electron microscope for molecular and cellular biology, Cryo-electron microscopy
Amount £2,340,000 (GBP)
Funding ID 206166/Z/17/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2016 
End 12/2018
 
Title Analysis of heterogeneity of samples 
Description Statistical methods for separation of molecular images according to their sizes or conformations 
Type Of Material Biological samples 
Year Produced 2009 
Provided To Others? Yes  
Impact Usage of the new tool by researches in structural biology using EM 
 
Title EM structural data 
Description Structural densitiy maps of portal proteins 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Densty maps for the core complexes 
 
Title Electron microscopy 
Description Analysis of the T4S systems in different organisms. The T4S systems are involved into active pathogenesis of many bacteria. We study the structural bases of the complexes to reveal a mechanism of thier activity. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Possible development of drugs, that would prevent pathogenic activity of bacteria and preventing of the horizontal transfer of genomic material between bacteria, a reason for their high resistance to antibiotics. 
 
Title Electron microscopy of biocomplexes using low and high end microscopes. Structural analysis of complexes form medium to high resolution. 
Description Firstly we are using cryo electron microscopy and automated data collection. Then we apply methods of image analyses such as determination of transfer function of the microscopes and statistical analysis of images, finally determination of orientations of particle images with three-dimensional reconstruction. That step is followed by interpretation of densities obtained during the reconstruction and analysis of variations in structures due to effects of the substrates. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact These methods allow us to improve a resolution of the EM structures and reveal conformational changes related to the function of the biocomplexes 
 
Title Flexible fittibng of atomoic structures 
Description Using Coot (X-ray software package), Phenix , and Em Flexible fit to make fitting of atomic models (both known X-ray structures and predicted based on homology modelling) . That allows us to make a reliable interpretation of bio complexes on the atomic level and to understand function-conformational changes that take place during proteins interactions 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact The methods is not absolutely unique, but it was essential to understand the function of the E1 heleicase and T4SS 
 
Title Image processing of EM micrographs 
Description Structural analysis of biological samples (proteins and macro biological complexes using digital approaches for alignment of images, their classification, determination of space orientation of the images and finally using methods of structural reconstructions of bio complexes in space. 
Type Of Material Biological samples 
Provided To Others? Yes  
Impact Approach has been implementsed in the package IMAGIC and broadly used in electron microscopy/ 
 
Title Structures of the T4SS complexes 
Description EM maps of densities 
Type Of Material Biological samples 
Year Produced 2010 
Provided To Others? Yes  
Impact Comparison of structure to do lableing of domains to deduct the way of action for the T4SS. 
 
Title EMDB 
Description This is a database (EMDB) that archives all structures obtained by electron microscopy (in negative stain and in cryo). All our results are deposited to this data base became available to other scientists 
Type Of Material Database/Collection of data 
Provided To Others? Yes  
Impact All our deposited data became available to the broad EM community and helps to analyse similarity and differences between bio complexes and investigate their functional activity. 
 
Title PDB 
Description The Protein Data Bank archive (PDB) serves as the single repository of information about the 3D structures of proteins, nucleic acids, and complex assemblies. It allows to validate structures and assess their quality using widely accepted standards and criteria. 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact That helps to us and other groups to make reliable interpretations of complexes and understand their functionality 
 
Description Analysis of the type 4 secretion system 
Organisation University College London
Department Division of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution With UCL (London) we do mass spec of samples purified in BBK, with TMSH (Houston, USA) we are working on a type 4 secretion system form agrobacteria. W are doing comparison of the T4SS complex from E coli system and Agrobacterium
Collaborator Contribution UCL did analysis of the molecular components with the the T4SS complex TMSH provided us the culture of bactria and biochemical information on the T4SS complex
Impact With UCL we were able to understand the way of oligomerisation of different protein components (PMID: 22745169) With TMSH we have running collaboration comparing two disccerent types of secretion systems from different bacteria . Both collaborations are multi disciplinary. we have got the first 3D reconstruction of the Agrobacterium T4SS. It is organisation seems to remind closely T4SS from E.Coli, but differences are observed in the inner membrane part.
Start Year 2008
 
Description Analysis of the type 4 secretion system 
Organisation University of Texas
Department Department of Microbiology and Molecular Genetics
Country United States 
Sector Academic/University 
PI Contribution With UCL (London) we do mass spec of samples purified in BBK, with TMSH (Houston, USA) we are working on a type 4 secretion system form agrobacteria. W are doing comparison of the T4SS complex from E coli system and Agrobacterium
Collaborator Contribution UCL did analysis of the molecular components with the the T4SS complex TMSH provided us the culture of bactria and biochemical information on the T4SS complex
Impact With UCL we were able to understand the way of oligomerisation of different protein components (PMID: 22745169) With TMSH we have running collaboration comparing two disccerent types of secretion systems from different bacteria . Both collaborations are multi disciplinary. we have got the first 3D reconstruction of the Agrobacterium T4SS. It is organisation seems to remind closely T4SS from E.Coli, but differences are observed in the inner membrane part.
Start Year 2008
 
Description EMBO cryo course 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Each time we have at least 80 applications from which we select 30 students, who attend the course. The scholl is internationally recognised and we have lectureres for the wolrd recogrnised laboratories

The schools have a strong educational impact. On the other side students (both PhD students and postdocs) extend communications with world recognised scientists and they are often interested in applying for jobs in London.
Year(s) Of Engagement Activity Pre-2006,2007,2009,2011,
 
Description EMBO cryo course on image processing 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Postdocs and PhD students were given lectures on principles of image analyses in electron microscopy. A number of world known scientists gave talks on their research and attendees had ana ample opportunity to talk to famous scantiest and to ask the about future plans and potential jobs.
Year(s) Of Engagement Activity 2015
 
Description GRK1721 (Integrated Analysis of Macromolecular Complexes and Hybrid Methods in Genome Biology) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach National
Primary Audience Undergraduate students
Results and Impact About 70 undergraduates and PhD students attended the talks and they were interested in the advanced technologies in structural analysis of biological complexes.

Students have gained knowledge on new tachnologies and approaches in structural biology
Year(s) Of Engagement Activity 2012
 
Description ISMB Retreat 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact More as 150 PhD students and posdocs from UCL and BIrkbeck college attend the retreat. Typically it has very engaging conversation between students and senior researches related to the future plans and possible activities within and nearby sciences.

Broadedning the mind a vision for students , to be reflected in their future plans both in scince and close to scince areas, like publications, editoral jobs etc.
Year(s) Of Engagement Activity 2008,2009,2011,2013,2014
 
Description IUCr 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Organising the future 2017 congress on crystallography. Considering potential key note speakers and sessions related both to electron crystallography and high resolution microscopy. Organising sponsorship to support of speakers and students and postdocs who are involved in research related to the electron crystallography.

It was an active discussion between the scientists involved in studies of materials by methods of electron crystallography. As chair of the Committee Electron Crystallography of the IUCr it was important to explain how the methodology developed in material sciences and biology can be merged, shared and developed for the mutual progress during international conferences, workshops and schools organised by the members of CEC.
Year(s) Of Engagement Activity 2014,2015,2016,2017
 
Description Open days in university 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact 400 people attended the event. During the open days I have been engaged into inter5sting conversation related to explanation of what is it structural biology, its role in medicine and education, improvement of environment.

Increasieng a number of undegraduates and graduates in BIrkbeck College
Year(s) Of Engagement Activity Pre-2006,2006,2007,2010,
 
Description Open days in university 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact more as 400 people attended the event

Increasing a number of undegraduates and graduates enrolloing to BIrkbeck College
Year(s) Of Engagement Activity 2006,2007,2008,2010,2013
 
Description Participation on Open days of Birkbeck college. 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Explanation of general ideas biolcogical studies, importance of structural studies and how it can be achieved. Making the links between microbiology, structural studies and development of means against diseases. Explanations of how the mutations in biological complexes can cause cancers and what we have to understand to be able to i=restore the functions of these molecules
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014