Toxic shock!

When a species other than c.botulinum was found producing its famous toxin – only the second report of a new botulinum toxin to be found in the past 40 years ­– one team needed to find out if the food chain was secure…

The discovery of the genes for making the world’s deadliest poison in bacteria living in the gut of a cow might sound like the beginnings of a major health scare, but in reality, it might point the way to new treatments for disease.

Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes botulism, a deadly neuroparalytic disease. It is the deadliest toxin known. If eaten, less than 1 ng/kg body weight is a fatal dose, and a teaspoon of the toxin would be enough to wipe out the entire population of the UK. If diagnosed quickly enough, most people can make a full, albeit slow recovery through treatment with antitoxin, but still around 5-10% of cases are fatal.

There are three main types of botulism. Wound botulism occurs when an open wound is infected with the bacteria, and is mostly associated with intravenous drug use. Infant botulism is caused by infection of the gut with bacterial spores. Young babies are susceptible to spore infection as they haven’t developed the defences against them, which we get from around 12 months old. The third and most prevalent cause is foodborne botulism, where someone consumes food containing the toxin. The presence of botulinum neurotoxin in food is a result of contaminating Clostridium botulinum spores being able to germinate, and the bacteria growing to a stage where they start to produce the toxin. Because of the severity of the consequences of this, much effort has gone into developing strategies that prevent foodborne botulism from happening, and modern food production incorporates stringent procedures and checks that now mean that foodborne botulism is very rare.

There are currently eight known botulinum neurotoxin serotypes and this is only the second report of a new botulinum toxin to be found in the past 40 years.

Developing these procedures has relied on an ever-increasing understanding of the biology of Clostridium botulinum. Here at the Quadram Institute (previously in the Institute of Food Research) we are proud to have played a major role in understanding the detailed life cycle of these bacteria. By studying what conditions are needed for the spores to germinate, and allow the bacteria to grow, reproduce, and make their toxin, we can help design procedures that prevent these things happening in the food chain.

We are continuing research efforts to study these bacteria. We can improve or modify food processing procedures based on new scientific knowledge to improve resource use efficiency, whilst still maintaining the same levels of safety in food production. We also need to be vigilant for new sources of outbreaks, or new strains of bacteria. And we can also look to use advances in scientific techniques to further our knowledge.

Deadly genes One of the most important advances for the study of C. botulinum, and many other pathogenic bacteria, has been the major advancements we have seen in genomics. We were part of a consortium, led by the Sanger Institute, that published the first genome sequence of a strain of C. botulinum in 2007. Comparative analysis of this genome uncovered many new insights into how the bacteria are adapted to their lifestyle, and how they form their highly potent toxin.

Given that it is the deadliest toxin, it’s not surprising that much study has gone into understanding the botulinum neurotoxin, including understanding from genomic studies the genes that the bacteria use to synthesise it. A cluster of genes is needed to make the toxin. As well as the gene for the toxin itself, there are a number of genes that encode accessory proteins that have a variety of functions ensuring that the toxin is correctly produced and deployed to its target cell.

From a genomics point of view, this also means that we can characterise the botulinum gene cluster not just from the genetic sequence of the toxin genes, but also from the close proximity of accessory protein genes.

We have now carried out a bioinformatic search of the National Centre for Biotechnology Information’s Whole Genome Sequence database for any entries that were similar to the predicted proteins that the botulinum toxin gene would produce. This produced a hit from a recently deposited bacterial strain whose botulinum neurotoxin shared 39% identical amino acid residues with its closest relative, BoNT/X (botulinum toxin type X), with 58% of the amino acid residues exhibiting conservative changes. The predicted product of this new neurotoxin gene contains all the structural domains expected for a botulinum neurotoxin; furthermore, its predicted 3D structure mimics that of the most potent botulinum toxin, BoNT/A (botulinum toxin type A).

Closer examination showed that there were genes upstream of this putative new neurotoxin gene that were similar to those encoding accessory proteins. We reported our discovery of the gene for a new botulinum neurotoxin in FEBS Letters (DOI: 10.1002/1873-3468.12969) and have designated the new botulinum neurotoxin eboNT/J.

Holy cow!

There are currently eight known botulinum neurotoxin serotypes (BoNT/A-G & X) and this is only the second report of a new botulinum toxin to be found in the past 40 years. All of the previous strains were found in Clostridium botulinum, or very closely related bacteria. Surprisingly, this new botulinum gene cluster was identified in a species of Enterococcus isolated from cow faeces in South Carolina.

The implications of this discovery now need to be considered. We don’t believe there is any major immediate threat to health from this. The cow wasn’t reported to show any signs of botulism. And as described earlier, C. botulinum is common in the environment. We would expect that the precautions taken to keep C. botulinum from causing problems in the food chain would be effective against any new strains of botulinum-producing bacteria, but this should be confirmed. It is an intriguing question as to how this Enterococcus strain acquired a botulinum neurotoxin gene cluster, and further work is required to explore the implications of our important finding with regard to the possibility of its transfer between bacteria.

Enterococcus bacteria typically inhabit the gastrointestinal tract of animals and humans. Some are commensals, making up a part of the normal microbiome that populates the gut. Others are known to cause disease.

The discovery of a novel type of botulinum toxin will also be of great interest to the pharmaceutical industry. Despite its disease-causing role, botulinum toxin is perhaps better known for its cosmetic use (e.g. Botox®). Botulinum neurotoxin blocks the transmission of neurotransmitters, causing muscles to relax. It is used cosmetically by targeting the stimulation of the tiny facial muscles that are behind wrinkles. Less well known are the therapeutic treatments which also exploit the neurotoxin’s ability to block nerve transmission. The constantly increasing range of medical procedures being treated with botulinum toxin include bladder dysfunction, multiple sclerosis, chronic migraine, severe axillary hyperhidrosis (underarm sweating), blepharospasm (involuntary eyelid closure), strabismus (misaligned eyes), cervical dystonia (involuntary contraction of the neck muscles) and upper and lower limb spasticity.

Each type of botulinum neurotoxin examined so far has different properties, for example potency and durability. The new neurotoxin (eBoNT/J) also shows features which suggest a novel target cell binding site. Once expressed as a protein, this new neurotoxin may possess novel properties, such as immunomodulatory properties making it useful for a very wide range of medical problems, for example, rheumatoid arthritis and Crohn’s disease and some cancers. It may also have properties that make it an ideal candidate for use as an alternative to existing botulinum neurotoxins such as Botox®. Determining the expression and toxicity of eBoNT/J will determine how it may be used as a therapeutic agent.

Discovering a new version of this deadly neurotoxin in a completely different species of bacteria is significant for the study of botulism, as it will provide new insights into the evolution and transmission of the genes, and maybe even into how the bacteria use their deadly weapon. But it’s also exciting to think that we may be able to turn this new weapon against some of the diseases where we are lacking effective cures at the moment.

We are a long way from realising this, and much more study will be needed, but this new discovery certainly adds to our armoury and demonstrates the power of genomic mining to uncover the unexpected.

 

Authors

Professor Mike Peck leads a research group at the Quadram Institute studying basic and strategic aspects of the physiology and molecular biology of Clostridium botulinum.  He holds Professorships at Nottingham University and the University of East Anglia, and has published more than 150 refereed articles and book chapters.

Dr Jason Brunt joined the Quadram Institute following a PhD in microbiology from Heriot-Watt University. He is currently a Research Scientist and QA Officer and his research focuses on the spore formers Clostridium botulinum and Clostridium sporogenes.

Dr Andrew Carter joined QI in 1990, initially as a yeast molecular biologist. For the last 13 years, he has studied the molecular biology of Clostridium botulinum in Prof. Mike Peck’s group.

Dr Sandra Stringer has worked in the QI Clostridium botulinum research group for 25 years. She has a degree in Food Science and a PhD in Food Microbiology, both from the University of Nottingham. Her research interests include the physiology and molecular biology of C. botulinum.