The Assembly of Tetrathionate Reductase in Pathogenic Bacteria

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

Bacteria are the simplest truly living things known to man. Some bacteria are dangerous and Salmonella is one of the primary causes of foodborne illness in the developed world. Many bacteria can live and grow without oxygen, and instead utilise other chemicals from the environment to generate energy for life. Salmonella is one such bacterium and it decorates the surface of itself with an impressive armoury of enzymes, which it uses to energize itself. Building these enzymes is a complicated process for the cell and involves the recruitment of metals atoms and other proteins to form the final finished enzyme. If scientists interfere deliberately in this process the new ?mutant? bacteria that are isolated are found to be no longer dangerous. The fact that many of these enzymes are often found outside on the surface of the cell is of key interest. How these enzymes get out the cell, and how they are fully assembled with all their metals and subunits attached before that, is the thrust of this research project. Most enzymes that are destined to be located outside the cell are identifiable by the presence of a special ?signal? on them. We have found this signal, which is also made of protein, has two jobs in the cell. First, it helps to assemble the subunits and metals, then second, it helps to locate the finished enzyme outside the cell. To do this the signal works in tandem with a ?chaperone? protein that interacts extensively with it and regulates its function. Once we learn in detail how these processes work we may be able to learn how design a new antibiotic to stop this system working in Salmonella without harming the environment. These types of processes are not used by human cells, which makes them a very attractive target for developing novel anti-bacterials.

Technical Summary

According to the Food Standards Agency, there are around 81,000 cases of foodborne illness annually in the UK. Salmonella species are some of the most common bacterial pathogens associated with foodborne illness in adults living in developed countries and are responsible for about 20-30% of food-related deaths. Salmonella is a Gram-negative bacterium and a member of the gamma-Proteobacteria that includes other human pathogens. The bacterium is a ?facultative anaerobe? and can thus use various compounds as replacements for oxygen during respiration. This has recently proven to be very important in the infection process. Experiments in a mouse model identified that the gut inflammation response, induced initially by Salmonella itself, causes the generation of tetrathionate in the gut mucosa through reaction of thiosulphate with reactive oxygen species. As Salmonella expresses a complex tetrathionate reductase enzyme, this tetrathionate can be used by Salmonella as a respiratory electron acceptor in order to out-compete the many other bacteria that lack this enzyme. The overall aim of this proposal is to provide fundamental information on the mechanism of targeting and assembly of tetrathionate reductase.

Tetrathionate reductase is a substrate of the twin-arginine translocation (Tat) pathway, which is a remarkable protein targeting system dedicated to the transport of fully folded proteins across the plasma membranes of bacteria. Indeed, animal pathogens can be rendered avirulent if their Tat systems are inactivated. Thus all aspects of this protein transport system are therefore of interest as potential future targets for novel anti-infectives. All proteins destined for export via the Tat translocase are synthesised with N-terminal twin-arginine signal peptides bearing conserved SRRxFLK amino acid motifs. The majority of Salmonella Tat substrates are cofactor-containing complex enzymes with key roles in respiratory electron transport chains. Pre-export assembly of such enzymes involves an important process that regulates access of the signal peptide to the translocase until cofactor loading and protein folding is complete. We call this process Tat proofreading and it involves the direct recognition and physical binding of the signal peptides by specific chaperones. This project involves a detailed study of the structure and function of paradigm members of the TorD family of Tat proofreading proteins, and their role in tetrathionate reductase assembly. By taking a multi-discipinary approach that combines structural biology, molecular biology, biophysics, microbiology, genetics, and the latest synthetic biology technology, we aim to shed light on the mechanism of signal peptide recognition by TorD-like proteins.

Publications

10 25 50
 
Description Structural biology of signal peptide binding proteins 
Organisation University of Dundee
Department Biological Chemistry and Drug Discovery (BCDD)
Country United Kingdom 
Sector Academic/University 
PI Contribution Protein production and characterisation. Molecular genetic studies and biomolecular interaction studies.
Collaborator Contribution Structural biology and x-ray crystallography.
Impact A multi-disciplinary paper was published in Biochemistry in Feb 2012 (Coulthurst et al. - listed in publications section). The work combines microbiology, molecular genetics, biochemistry, crystallography and structural biology.
Start Year 2011
 
Description Dundee Doors Open 2012 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact More than 200 people visited the College of Life Sciences on Saturday November 10, 2012. Different instruments and stalls were on display, including BBSRC-funded instrumentation. There were questions and dicussions.

Unknown.
Year(s) Of Engagement Activity 2012
 
Description Magnificent Microbes 2012 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a two-day event at Dundee Science Centre. On day one (a Friday), 180 primary school prupils attended. On day-two 600 members of the public attended. There were several interactive stalls educating on many different aspects of microbiology. There were no stuffy presentations from boring old professors. It was fun, interactive and informative.

Interest from the local media (The Courier newspaper) and from BBSRC public engagement officers.
Year(s) Of Engagement Activity 2010,2012
 
Description Magnificent Microbes 2014 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This was a two-day event designed by academics and staged at Dundee Science Centre. Several interactive stalls were set up and these were used to stimulate discussions with the visiting public. The first day involved visits from selected primary schools from the East of Scotland. The second day was open to the public.

School children and their teachers were surveyed before and after the event on their knowledge and understanding of microbiology. There was a notable shift afterwards.
Year(s) Of Engagement Activity 2014
URL http://www.lifesci.dundee.ac.uk/impact/schools-outreach/media/magnificent-microbes-2014