Bacterial type III secretion systems: from structure to function

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.

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.

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.