Exploiting the structure of the Type 9 Secretion System protein translocon

Secretion of proteins into the environment is an fundamental feature of bacterial life. This is the mechanism by which pathogenic bacteria cause disease. Likewise, the bacteria that break down plant fibres into biofuels must secrete enzymes to digest the fibres because the fibres are too big to be transported into the bacterium.
The Bacteroidetes are an important group of bacteria found in the digestive tracts of animals and humans, as well as being widespread in the environment. Recent research has shown that Bacteroidete bacteria use a previously-undiscovered apparatus to export proteins, called the type 9 secretion system, or T9SS for short. The T9SS is essential for dental pathogens to cause perditontal disease and to cause the main bacterial diseases of salmon and other farmed fish.
We have recently isolated the pore in the bacterial cell surface through which the T9SS secretes proteins and have determined its structure using electron microscopy. We will use this structural information as a basis for experiments to understand how the T9SS pore operates. We will also investigate how the pore collaborates with other components of the T9SS to achieve protein export from the cell.
This work has the potential to aid the design of drugs to attack pathogenic Bacteroidetes as well as to help us understand how to exploit other Bacteroidetes to make biofuels from plant fibres.

We recently succeeded in identifying the core outer membrane protein translocon of the Type 9 Secretion System (T9SS). We have used cryo-electron microcsopy (cryo-EM) to determined the 3.6A structure of this protein complex in two states. We now aim to exploit this breakthrough to determine the mechanism of T9SS transport through:

(1) Structure-informed tests of core translocon mechanism. Our structures suggests plausible mechanistic models for the transport of the substrates through the T9SS translocon. We will biochemically test these models taking advantage of the molecular level structural context we now have available to guide our experimentation. Specifically we aim to:
- test the function of T9SS components that were identifed for the first time from the translocon structures.
- examine conformational dynamics in the translocon through site-specific disulfide crosslinking.
- test a proposed subunit exchange using a live cell imaging approach.
- validate the proposed protein transport pathway through the translocon.
These experiments are expected to allow us to elucidate the general mechanism of the T9SS core translocon.

(2) Determining structures of the translocon in different stages in the transport cycle. This work will take advantage of the fact that the translocon is an exceptionally well-behaved cryo-EM target. Specifically we aim to obtain structures of the T9SS translocon in complex with:
- the T9SS-targeting domains of substrate proteins.
- other components of the T9SS pathway that associate with the translocon.

The Type 9 Secretion System (T9SS) is required for environmental and rumen bacteria of the phylum Bacteroides to break down solid carbohydrate substrates (such as plant fibres). It is also essential for the virulence of pathogenic Bacteroidetes including those responsible for human peridontis, the major bacterial diseases of farmed fish (including columinaris disease, rainbow trout fry syndrome, and cold-water disease), and some avian diseases. It is further implicated in the maintenance of beneficial root commensals.
The aim of the work described here is to provide fundamental information on the mechanism of T9 secretion. However, our results will be relevant in underpinning commercial efforts to exploit the T9SS through
- use of Bacteroidetes to break down complex solid carbohydrates for biofuel production.
- improving our understanding of basic bacterial cell biology relevant to animal and human pathogenesis.
- characterizing a target for novel antimicrobials.
The work, therefore, has relevance to the BBSRC Strategic Priorities `Bioenergy; generating new replacement fuels for a greener, sustainable future' and 'Animal Health',
In addition there may be relevance to the BBSRC Strategic Priority 'Integrative microbiome research' since the T9SS is part of the network for interactions betwen bacteria in both animal and plant commensal settings.
Although the proposed project does not encompass the development of antimicrobial compounds per se, CI Lea has expertise in structure based drug design and so we are equipped to recognise and highlight those results of our work with promise in this area.
Communication with potential industrial beneficiaries will take place via the technology transfer infrastructures of the University of Oxford. Specifically, we will patent intellectual property arising from this research, and then seek to license or spin-out this technology with the support of Oxford Innovation Ltd.
The primary mechanism for communication of this research will be through publication in peer review international journals and in accordance with Research Council Open Access policy. We will liaise at the time of publication with the University of Oxford and BBSRC 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.