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.
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.
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.
-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.
Organisations
- University of Bristol (Lead Research Organisation, Project Partner)
- ETH Zurich (Collaboration)
- MRC-Technology (Collaboration)
- Creative Antibiotics (Collaboration)
- Microbiotix Ltd (Collaboration)
- University of Bristol (Collaboration)
- Kennesaw State University (Project Partner)
- Osaka University (Project Partner)
- University of Utah (Project Partner)
- Umeå University (Project Partner)
Publications
Cheung M
(2013)
A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy.
in Journal of structural biology
Marten F
(2012)
Bacterial secretion and the role of diffusive and subdiffusive first passage processes.
in PloS one
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
Perdu C
(2015)
ExsB is required for correct assembly of the Pseudomonas aeruginosa type III secretion apparatus in the bacterial membrane and full virulence in vivo.
in Infection and immunity
Murillo I
(2016)
Genetic Dissection of the Signaling Cascade that Controls Activation of the Shigella Type III Secretion System from the Needle Tip.
in Scientific reports
Martinez-Argudo I
(2013)
Isolation of Salmonella mutants resistant to the inhibitory effect of Salicylidene acylhydrazides on flagella-mediated motility.
in PloS one
Shen DK
(2016)
MxiA, MxiC and IpaD Regulate Substrate Selection and Secretion Mode in the T3SS of Shigella flexneri.
in PloS one
Shen DK
(2012)
Needle length control and the secretion substrate specificity switch are only loosely coupled in the type III secretion apparatus of Shigella.
in Microbiology (Reading, England)
Verasdonck J
(2015)
Reassessment of MxiH subunit orientation and fold within native Shigella T3SS needles using surface labelling and solid-state NMR.
in Journal of structural biology
Roehrich AD
(2013)
Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation.
in Molecular microbiology
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 | Public |
Country | United Kingdom |
Start | 05/2016 |
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 | 05/2015 |
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 | 05/2013 |
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 | 06/2013 |
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 | Public |
Country | United Kingdom |
Start | 09/2011 |
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 | 05/2013 |
End | 10/2014 |
Description | WT Undergraduate Student Vacation Scholarship |
Amount | £2,000 (GBP) |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2013 |
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 | 03/2015 |
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 | 05/2009 |
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 |
Country | United States |
Sector | Private |
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 | Private |
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 |