Triggering assembly of the twin-arginine translocase

All bacteria produce proteins that operate on the outside of the bacterial cell. Good examples of this are the toxins produced by bacterial pathogens. Since bacteria only make proteins inside the cell, the newly-synthesised extracellular proteins must be moved out of the cell across the normally impermeable cell membrane. This task is carried out by machines termed protein transporters that are located in the cell membrane.

The Tat protein transporter is found in many different bacteria including almost all those which cause human diseases such as Mycobacterium tuberculosis (which causes tuberculosis), and Salmonella (which causes food poisoning). In all pathogenic bacteria tested the Tat transporter is essential for the bacterium to cause disease. Thus, scientists are becoming increasingly interested in developing drugs that prevent the Tat system from working.

In order to understand how the Tat machinery works we will use cutting edge molecular modelling, dynamics and experimental approaches to elucidate how the transport machinery recognises the targeting tags present on the proteins that are to be transported. This knowledge will help underpin attempts to understand and inhibit this pathway in pathogens.

The twin arginine protein transport (Tat) system is a conserved and highly unusual protein transport pathway that exports folded proteins across the cytoplasmic membranes of bacteria. It is required for the virulence of most important human pathogens and is essential for the viability of pathogenic mycobacteria. The Tat system assembles 'on demand' in the presence of a substrate protein and disassembles once transport is completed. The aim of this study is to provide key information about the interaction of Tat substrates with the Tat receptor through a combination of molecular dynamics, modelling, in vivo crosslinking and biochemistry. It has the following specific objectives:

1. To map the signal peptide binding site within the Tat receptor and to determine whether binding of the signal peptide at this site is dependent on the protonmotive force;

2. To determine whether the newly-discovered TatA/B binding site on TatC TM6 is essential for operation of the Tat pathway and;

3. To examine the organisation and activation mechanism of the Tat receptor in Tat translocases that lack the TatB component.

These data will provide crucial information on how the substrate protein interacts with the Tat system during the translocation cycle. This fundamental knowledge will support drug discovery activities and underpin commercial efforts to exploit the Tat pathway in the production of proteins of therapeutic relevance.

The export of proteins by bacteria is a critical process that is essential for their survival and for the delivery of virulence factors during pathogenesis. The Tat pathway is a highly unusual protein transport system that exports folded proteins across the bacterial cytoplasmic membrane. The Tat system is found in most pathogenic bacteria (including the genera Salmonella, Escherichia, Shigella, Yersinia, Legionella, Vibrio, Helicobacter, Pseudomonas, Mycobacterium,
Staphylococcus, Bacillus, Streptococcus, Haemophilus, Brucella, Campylobacter, Rickettsia, Bordetella, Burkholderia, Neisseria, and Klebsiella) and has been found to be essential for virulence in all tested cases. The Tat system is an
essential pathway in some bacteria including Mycobacterium tuberculosis, the causative agent of tuberculosis. Since the Tat system is absent from human cells it represents a novel target for development of antibacterial compounds and several groups are actively screening for small molecules that interfere with the operation of the Tat pathway.

The aim of the work described here is to provide fundamental information on the mechanism of Tat transport. This fundamental knowledge will support drug discovery activities. It is also relevant in underpinning commercial efforts to
exploit the ability of the Tat pathway to transport folded proteins in the production of proteins of theraputic relevance. Communication with potential industrial beneficiaries will take place via the technology transfer infrastructures of the
University of Newcastle and Oxford. Specifically, we will patent intellectual property arising from this research, and then seek to license or spin-out this technology.

The primary mechanism for communication of this research will be through open access publication in peer review international journals. We will liaise at the time of publication with the University of Newcastle and Oxford Press offices and the MRC to ensure publicity of results of interest to the general public. Our results will also be made available on our regularly updated web sites. Note also that the Tat system is now featured in mainstream cell biology text books such as Molecular Biology of the Cell and so our data will potentially impact on future editions of standard texts.

The researchers employed on this grant will gain technical skills in cutting edge methodology in bacterial molecular genetics, cell biology, molecular dynamics and molecular modelling and in the application of such techniques in
complex systems involving integral membrane proteins. The biophysical parts of the study will provide a training in quantitative data analysis and the researchers will also gain writing, IT, and presentational skills. Researchers in our
laboratories take part in Departmental Science Open Days (typically putting on practical demonstrations in protein science or bacteriology).