Bacterial type III secretion systems: from structure to function

Lead Research Organisation: University of Bristol
Department Name: Cellular and Molecular Medicine

Abstract

Many pathogenic Gram negative bacteria possess tiny cell surface-localised injection devices, called type III secretion systems (T3SSs), dedicated to the delivery of virulence- mediating proteins into the cells of their eukaryotic hosts. Through the distal tip of their injection needle, these devices directly sense physical contact with host cells and activate the secretion machinery within the bacterium. We wish to understand how this initial cross-talk, which represents a fascinatingly complex signal transduction event between two very different types of organism, occurs at the mechanistic and molecular level.

Our work will directly help understand how bacteria such as Shigella, Salmonella, Pseudomonas, Chlamydia and Yersinia, human pathogens which all carry T3SSs, first interact with the cells of their host. Some of the proteins involved are the only known protective antigens against these organisms, and their in-depth study may generate new opportunities for vaccine design. Precisely defining their molecular roles may also lead to the development of therapeutic drugs. Antibiotic resistance is ever increasing and spreading rapidly between different bacterial species, while fewer and fewer chemically new types are being discovered. The rational design of new anti-microbial drugs can be initiated from screening chemical libraries but this first requires that appropriate biological targets are defined. This can only be achieved by increasing basic mechanistic knowledge of how specific and conserved virulence factors operate. This is precisely what we propose to do.

T3SSs carrying-bacteria are a major cause of infectious diseases not only in humans, but also in animals and plants, even in the developed world. As T3SS are so wide-spread and conserved, what we find may be applicable to preventing/treating numerous diseases in humans, animals and crops plants.

Technical Summary

Aim I -Define entire structure of Shigella T3SS:
a) Obtain near-atomic resolution 3D reconstruction of needle and selected mutants by cryoEM;
b) Produce 5-9 Å resolution non-symmetrised 3D map of NC by cryoEM (including mapping location & orientation of CIMEA components, integrating views of needle & TC into single high-resolution view of wild-type NC and reconstruction of mutant NCs);
c) Obtain tomographic reconstruction of whole T3SS within intact bacteria.

Aim II -Define path taken by activation signal from TC until late effector secretion:
a) Screen for mutants altered in activation throughout NC (using already identified signal-insensitive mutants in TC components to initiate a colony colour-based suppressor screen);
b) Progress towards holistic understanding of regulation of T3SS substrate expression by generating an RNA map of Shigella virulence plasmid;
c) Progress towards understanding mechanisms mediating secretion hierarchy by quantifying secretion processes
(both of the latter objectives will be executed in different mutants and under conditions representing different stages in T3SS activity);
d) Examine location of export substrates and their mRNAs relative to T3S machineries, as well as diffusive properties of the former to understand whether they contribute to secretion hierarchy establishment.

Aim III -Build tools to reconstitute export of T3 substrates across the inner membrane in vitro:
For technical reasons we will here use the Salmonella flagellar T3SS as a model system to
a) Generate & test mutants and genetic constructs for monitoring export activity in a strain overexpressing flagella;
b) Generate, affinity purify and characterise inverted inner membrane vesicles (IMVs) derived from above strain;
c) Prepare cytosol and substrate (s), mix with IMVs and an energy source and monitor export activity enzymatically or radioactively;
d) Perform initial functional characterisation of in vitro assay.

Planned Impact

Beyond our academic colleagues, potential beneficiaries of our research include:
-the Public Sector, given that we study the agent of bacillary dysentery in humans for which there is still no vaccine available and where the T3SS surface components are strong diagnostic and protective antigen candidates. This includes, for instance, international organizations such as the International Vaccine Institute (Seoul, Korea), The Global Alliance for Vaccines and Immunisation and national vaccine development organizations such as the US National Institute of Child Health and Human Development and the UK Jenner Institute.

-the commercial Private Sector, whether biotechnology start-up (Creative Antibiotics, Mutabilis, Biotics) or large Pharmaceutical (Novartis/GSK) companies, given that there is renewed demand, and now international charity funding (via the Bill & Melinda Gates Foundation, the International Finance Facility for Immunisation, Advanced Market Commitments for Vaccines), for vaccines against diarrheal diseases, as well as for new "anti-virulence" drugs to combat the ever increasing antibiotic resistance of bacterial pathogens. Since the T3SSs portions we propose to study are the most conserved part of these apparatuses, which are broadly distributed amongst bacterial pathogens of humans, animals and plants, our work has a strong impact potential.

- the general public, who is interested in how micro-organisms cause disease, and how we can combat them particularly -in the developing world- and also in debates on Evolution in which our work is often used as evidence.

The timescale and depth in which these sections of society are likely to benefit vary enormously although we will to ensure that these are optimized to the best of our ability.

We aim to publish our work in high profile journals, so that our findings are easily accessed by pharmaceutical researchers. AJB is involved in collaborations with a start-up company and interacts with several larger pharmaceutical companies, which will allow us to present our research to the industry, and pursue its potential for drug development.

We will engage with the public, by taking part in activities such as "junior science cafes" in schools. In addition, we actively encourage lab members to take part in other public engagement activities within schools, such as the "researchers in residence" scheme. The public can also access our research via our School's webpages. In future, we would like to participate in events aimed at explaining the basics of infectious diseases to children at "@Bristol", which is a local hands-on interactive science centre. Finally, the University of Bristol houses the Institute for Advanced Studies, which has a commitment to public engagement in science as well as involvement in establishing the National Co-ordinating Centre for Public Engagement (hosted jointly with The University of The West of England). Through this a number of activities are arranged such as talks at local schools, involvement in summer school programmes, interaction with the media and events to increase public understanding of science.

Publications

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Fujii T (2012) Structure of a type III secretion needle at 7-Å resolution provides insights into its assembly and signaling mechanisms. in Proceedings of the National Academy of Sciences of the United States of America

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McKetterick TJ (2014) Exact dynamics of stochastic linear delayed systems: application to spatiotemporal coordination of comoving agents. in Physical review. E, Statistical, nonlinear, and soft matter physics

 
Description BristolBridge (project title: Early phase development of an AFM-based primary care device to detect rapidly antibiotic resistance in common bacteria)
Amount £18,500 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 08/2016
 
Description Catalyst Award from Elizabeth Blackwell Institute at the University of Bristol (project title: Rational vaccine design: can one identify protective antigens systematically in silico? A pilot study focusing on epitope design for Shigella and Salmonella)
Amount £50,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2015 
End 08/2016
 
Description GS Undergraduate Student Summer Vacation Scholarship
Amount £2,000 (GBP)
Organisation The Genetics Society 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 08/2013
 
Description Hosted rotation of a PhD student from Bristol Centre for Complexity Sciences
Amount £3,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2013 
End 09/2013
 
Description Shared PhD student from University of Bristol Faculties of Med. and Dent. and Med. and Vet. Sci. MRC Doctoral Training Grant
Amount £95,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description TRACK award from the Elisabeth Blackwell Institute at the University of Bristol
Amount £15,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 10/2014
 
Description WT Undergraduate Student Vacation Scholarship
Amount £2,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2013 
End 09/2013
 
Description Wellcome Trust New Investigator Award WT104634AIA
Amount £750,000 (GBP)
Funding ID WT104634AIA 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2019
 
Description Wellcome Trust Project grant WT088231 A high-resolution mass spectrometry and electron microscopy approach to understanding the function of the Shigella T3SS
Amount £473,686 (GBP)
Funding ID WT088231 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2009 
End 03/2013
 
Title 20 A electron density maps of the T3SS distal needle tip complex and docked pseudoatomic model of IpaD tretramer 
Description The EM maps and pseudoatomic model were deposited in the Electron Microscopy Data Bank and the Protein Data Bank under accession numbers EMD-2801, EMD-2802, EMD-2803, EMD-2804, EMD-2806, EMD-2805 and 4D3E, respectively. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2014 
Provided To Others? Yes  
Impact Too early to say 
 
Title 7A resolution electron density map of the Shigella T3SS needle 
Description The EM map and a 22-mer pseudoatomic needle model were deposited in the Electron Microscopy Data Bank and the Protein Data Bank under accession numbers EMD-5352 and 3J0R, respectively. 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact Comparison with other research data such as the following paper: Atomic model of the type III secretion system needle. Loquet A, Sgourakis NG, Gupta R, Giller K, Riedel D, Goosmann C, Griesinger C, Kolbe M, Baker D, Becker S, Lange A. Nature. 2012 May 20;486(7402):276-9. doi: 10.1038/nature11079. Erratum in: Nature. 2012 Aug 30;488(7413):684. Please see also associated corrigendum: Loquet A, Sgourakis NG, Gupta R, Giller K, Riedel D, Goosmann C, Griesinger C, Kolbe M, Baker D, Becker S, Lange A. Nature. 2012 Aug 8. doi: 10.1038/nature11350. and subsequent publication: High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy. Demers JP, Habenstein B, Loquet A, Kumar Vasa S, Giller K, Becker S, Baker D, Lange A, Sgourakis NG. Nat Commun. 2014 Sep 29;5:4976. doi: 10.1038/ncomms5976. 
URL http://www.ebi.ac.uk/pdbe/entry/EMD-5352
 
Description Collaboration with Microbiotix on novel small molecule T3SS inhibitors 
Organisation Microbiotix Ltd
PI Contribution Tested small molecule inhibitors of Pseudomonas T3SS in our assays for functionality of Shigella T3SS.
Collaborator Contribution Provided selection of small molecules
Impact Small molecules selection did not work on Shigella.
Start Year 2014
 
Description Collaboration with ssNMR spectroscopists to resolve controversy over high resolution structure of T3SS needle 
Organisation ETH Zurich
Country Switzerland 
Sector Academic/University 
PI Contribution We are preparing native T3SS needles labelled with 15N and 13C for solid state NMR
Collaborator Contribution They are collecting the data (via the BioNMR EU project) and analysing it with us.
Impact This is evidently multi-disciplinary since our colleagues are biophysical physicists.
Start Year 2013
 
Description Identification of molecular target of small molecule T3SS inhibitors 
Organisation Creative Antibiotics
Country Sweden 
Sector Private 
PI Contribution We studied the effects of the compounds on Shigella flexneri and Salmonella typhimurium T3SS-related systems and have published one paper on the Shigella work already (PMID: 18996990), whilst the Salmonella work (PMID:23300965). Thus, this work in an indirect consequence of having MRC funding to support my lab in general. It paid for some of the consumables used by an EEC funded postdoc and a technician to do the work and it also supported part of the salary of the part-time technician.
Collaborator Contribution Company gave inhibitory compounds to be tested.
Impact We believe we have identified the targets of the these drugs using a genetic screen and whole genome sequencing. This work is now published in PLoS One.
Start Year 2007
 
Description Rational design of peptide inhibitors of T3SS activation 
Organisation MRC-Technology
Country United Kingdom 
Sector Academic/University 
PI Contribution Basic research underlying virulence mechanism to be targeted. ELISA based T3SS induction assay and technical personnel time.
Collaborator Contribution Assistance with design of peptides to be tested. Purchase of peptides to be tested. Synthesis, purification and characterisation of some peptides. Assistance with project progress through MRCT assessment and development scheme.
Impact Inhibitors did not work
Start Year 2012
 
Description Rational design of peptide inhibitors of T3SS activation 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution Basic research underlying virulence mechanism to be targeted. ELISA based T3SS induction assay and technical personnel time.
Collaborator Contribution Assistance with design of peptides to be tested. Purchase of peptides to be tested. Synthesis, purification and characterisation of some peptides. Assistance with project progress through MRCT assessment and development scheme.
Impact Inhibitors did not work
Start Year 2012
 
Title Peptide-based inhibitors of T3SS assembly or function 
Description Presently confidential. Being performed with assistance of MRC Technology and Wellcome Trust funding from the Elisabeth Blackwell Institute at the University of Bristol. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2013
Development Status Actively seeking support
Impact Not applicable yet 
 
Title Bespoke programmes for 3D reconstruction of T3SS distal needle tip complex 
Description Set of programmes, instructions and test data sets released via MRC funded CCPEM and University of Bristol Data Repository (10.5523/bris.1trtuj35bwn5410ibhinps47ag) 
Type Of Technology Software 
Year Produced 2014 
Open Source License? Yes  
Impact Too early to say 
URL http://www.ccpem.ac.uk/download.php
 
Company Name Creative Antibiotics 
Description Creative Antibiotics is developing virulence blockers which can be alternative or complementary to current antibiotics. Currently, Creative Antibiotics has three projects: agents against infections in severe burns, agents against diarrhoea and agents against chlamydia infections. http://www.creativeantibiotics.com/default.asp?LID=1 
Impact Tried to use our understanding of T3SS distal needle tip complex to help company design better PcrV inhibitors. Unfortunately, they went out of business in June 2013.
Website http://www.creativeantibiotics.com/default.asp?LID=1
 
Description School visits-South Gloucestershire 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Local
Primary Audience Schools
Results and Impact ~10 International Baccalaureate students attended lecture by PI as a supplement to their Biology course.
Postdoc ran a session on basic Microbiology in a primary school.
PI ran session on Infectious Diseases using Hollywood film "Contagion" for year 7-8 children.

Schools asked us back and we are now visiting other schools locally.
Year(s) Of Engagement Activity 2008,2009,2013