Towards a molecular, mechanistic, understanding of bacterial type three secretion

Secretion of proteins into the environment is a fundamental feature of bacterial life. This mechanism allows pathogenic bacteria to cause disease both by secretion of toxins and also by assembling the secreted proteins to form the nano-machines required for motility. Many important human pathogens use type 3 secretion systems, T3SS for short, to perform these functions with deletion of the T3SS rendering the bacteria non-pathogenic. The T3SS is particularly important for eight of the twelve bacterial families highlighted by the World Health Organisation as those for which we critically need to develop novel antibiotics due to their world-wide disease burden.
T3SSs assemble a large nano-machine and work arising from our earlier Programme Grant has recently determined the structure of the gate that controls secretion and lies at its core. This structure has revealed an unexpected location for the gate within the assembled machine that now requires significant redrawing of our previous hypotheses about how T3SS function. This structural information forms the basis for experiments to understand how T3SSs operate. In particular we want to understand how the nano-machine operates to push the cargo out of the bacteria, though the oily membrane that defines the organism. This process requires energy, but it is currently completely opaque how that energy is used to achieve export of precisely the right proteins. By determining molecular structures we hope to be able to gain insight into this critical process by understanding how the molecules involved are arranged and re-arranged to achieve the export step.
This work has the potential to aid the design of drugs to attack pathogenic bacterial families by providing information about molecular detail of the mechanics of this nano-machine that may suggest novel routes to break the system using drug-like molecules.

We seek to determine the structures of mechanistically informative sub-complexes of the entire type 3 secretion machinery in functionally informative states to design mechanistic hypotheses to be tested in vivo. We will use a combination of high resolution single particle cryo-EM and X-ray crystallographic studies as our main structural methodologies. These methodologies are well established in the lab as demonstrated by the prior publications and preliminary data supporting this application. Many of the protein complexes to be studied are integral membrane complexes and we will study these either in detergent or SMA peptide extracted forms and image in either detergent micelles or lipid nanodiscs with our choices of extraction and purification regimes guided by native mass spectrometry to characterise complex composition, heterogeneity and stability. This approach was crucial to our recently determined structure of the export gate complex that provides the strong start point for further studies designed to understanding the process of substrate selection and gating.
Specific initial targets are founded on strong preliminary data that suggest these are likely to be tractable over the five year timescale requested and include:
1. Structure of a FliPQRB complex at a resolution sufficient to allow us to build the structure of the unknown N-terminal portion of B and gain insight into this critical component.
2. Structure of full-length FlhA as found in vivo either by determining the structure of the nonameric complex or by determination of the structure of the monomer and modelling of the nonamer into existing tomographic data
3. Structure of the C-ring/pod-ATPase complexes by determination of structures of sub-complexes of the full assembly and modelling into tomographic data of the full assembly.
4. Structures of the Mot complexes that power flagellar rotation using ion-flow
5. Structures of the non-flagellar host-cell pore

The Type 3 Secretion System (T3SS) is a key determinant of pathogenicity for many human, animal and plant pathogens including those responsible for dysentery and plague. Of the World Health Organisation list of 12 bacterial families for which antibiotics are urgently required (http://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed) eight are dependent on T3SS to cause disease.
The aim of the work described here is to provide fundamental information on the mechanism of T3 secretion. However, our results will be relevant in underpinning efforts to target the T3 in:
- improving our understanding of basic bacterial cell biology relevant to animal and human pathogenesis.
- characterizing a target for novel antimicrobials.
The burden imposed by T3SS bearing bacteria for global mortality and morbidity has relevance to the MRC Strategic Priorities `Picking research that delivers: Natural protection' and 'Going global: Global health'
Although the proposed project does not encompass the development of antimicrobial compounds per se, we have expertise in structure-based drug / vaccine design and so are equipped to recognise and highlight those results of our work with promise in this area. We will patent any appropriate intellectual property arising from this research, and then seek to license this technology with the support of Oxford University Innovation Ltd.
The primary mechanism for communication of this research will be through publication in peer review international journals in accordance with Research Council Open Access policy. We will liaise at the time of publication with the University of Oxford and MRC Press offices to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated departmental web sites.
The researchers employed on this grant will gain technical skills in cutting edge methodology in protein chemistry, genetics, cryo-electron microscopy, and X-ray crystallography, and in the application of such techniques in complex systems involving integral membrane systems. The researchers will also gain writing, IT, and presentational skills. Researchers in the PI's group at Oxford are expected to take part in Departmental Science Open Days and other public events (typically putting on practical demonstrations in protein science or bacteriology) and in the PI's science outreach activities at local schools.