Please can you give a brief introduction to the cholera toxin and explain how it attacks the cells of the intestine?
Cholera bacteria, and other types of bacteria that cause diarrheal diseases, infect your intestines where they release AB5 protein toxins – that is they have a single toxic A-subunit that is linked to a pentamer of B-subunits that act as the delivery vehicle to transport the A-subunit into the cells.
The B proteins stick to a specific carbohydrate called GM1 on the surface of intestinal cells and the binding event leads to internalisation of the toxin.
It was recently announced that you have created an inhibitor that prevents the cholera toxin from binding to intestinal cells. How was this developed?
As the B-subunit has five sites that can interact with the target sugar, you need to block all five sites to stop the toxin from being internalised.
One strategy to achieve this is to make a multivalent inhibitor – that is one which has multiple copies of the ligand attached to a scaffold molecule.
What prompted you to use an inhibitor based on cholera toxins? Was this a case of “fighting fire with fire”?
Ideally you want an inhibitor scaffold that would match the number of sugars groups with the number of binding sites on the protein, and that the scaffold would also match the size of target protein.
In the case of cholera toxin, the binding sites are about 3 nm apart. It is very challenging to make well defined synthetic molecules of that size, but we realised the protein we were targeting was itself the correct size and shape, so why not use that as the scaffold on which to display the sugar ligands? Yes, you could say it is fighting fire with fire.
How exactly does the inhibitor work?
The first thing we did was to make a mutation in the gene for the cholera toxin B-subunit to make a mutant protein that was unable to stick to its natural sugar ligand: the result was an inert protein that we could use as a scaffold on which to attach five copies of a synthetic sugar ligand.
This was achieved by selectively oxidising one amino acid in each protein chain and then chemically attaching the sugar ligand to the protein.
The resulting inhibitor makes a complex with the toxin protein in which each of the five appended sugar groups binds to a different site on the pentameric toxin’s B-subunit. If any one of these sugars disengages from the toxin, the other sugars continue to hold on the inhibitor which increases the chance the disengaged sugar will rebind. Therefore, once the complex has formed it is very stable.
What were the main benefits of using an inhibitor based on cholera toxins?
The cholera toxin B-subunit can be manufactured on an industrial scale which means that production of the inhibitor could potentially be scaled up.
It also allows the formation of discrete complexes rather than large aggregates which form with other types of multivalent inhibitors. In some circumstances aggregates could be almost as harmful as the parent toxins.
Were there any potential downsides you had to bear in mind?
Protein-based drugs are becoming very common in the clinic, but they do have a disadvantage of being quite expensive to manufacture. This means that a drug based on our inhibitors may well be too expensive for treating cholera in developing countries.
However, there are very similar types of toxins produced by E. coli O157 which enter the blood stream and attack kidney cells causing haemolytic uremic syndrome. Using a protein-based inhibitor of that toxin could be a very viable treatment during an outbreak of food poisoning.
Where can readers find more information?
The paper we published is open access which means anyone can download it for free from the Angewandte Chemie website (http://onlinelibrary.wiley.com/doi/10.1002/anie.201404397/abstract), as is a review we wrote about inhibitors of bacterial toxins (http://pubs.rsc.org/en/Content/ArticleLanding/2013/CS/c2cs35430f#!divAbstract).
You can also find out more information about the importance of carbohydrates in biology and medicine from this video on YouTube https://www.youtube.com/watch?v=DHCpbEZ3kEg.
About Dr Bruce Turnbull
Dr Bruce Turnbull is an Associate Professor in the School of Chemistry and Astbury Centre for Structural Molecular Biology at the University of Leeds. He was awarded the 2013 Royal Society of Chemistry prize for carbohydrate chemistry. He chairs a European COST Action Network called Multivalent Glycosystems for Nanoscience which involves over 60 research groups from 21 countries. Their aim is to exploit protein-sugar interactions to develop new drug delivery systems, medical diagnostics, vaccines and inhibitors of bacteria and viruses.
Targeting skin microbiome rivals: Unveiling epilancin A37's antimicrobial mechanism