By: Juliet Greenwood ‘21 and Rebecca Marsden ‘20
The name Clostridium botulinum may ring a few bells. This microbe and its stunting mechanisms play many opposing roles in our world. The bacterium is responsible for producing the lethal neurotoxin that causes botulism. Botulism is a disorder of neuroparalysis in vertebrates that does permanent damage to the nervous system. It most often poses a threat to humans when it contaminates foods like improperly canned goods or home-made alcohols. If left untreated, it can be fatal (CDC 2018). Its dangerous toxicity and resistant spores also place it into the category of a potential agent of biowarfare. On the flip side, Clostridium botulinum has a starring role in onabotulinumtoxinA treatments, more commonly known as BOTOX. This cosmetic treatment involves a voluntary injection of the neurotoxin into face muscles to prevent wrinkles from forming.
Figure 1. Comedic cartoon depicting the various roles of the botulinum neurotoxin by Mark Parisi.
While Clostridium botulinum is notorious both in the food and pharmaceutical world, the bacteria exists abundantly in nature as a spore-forming soil microbe. It falls into the category of anaerobic saprophyte, meaning it thrives in low-oxygen decaying environments like intestines, soil, or fermenting foods. The bacteria itself is rod-shaped, but takes on a club shape when it forms prepackaged dormant spores (Figure 2). What you can’t see under the microscope is its neurotoxins; proteins which cut off communication between the central nervous system and muscles at a meeting point called the neuromuscular junction [1].
Figure 2. Clostridium botulinum, rod-shaped and club-shaped spore-forming bacteria.
Clostridium botulinum is only one member of a large family of bacteria; the genus Clostridia. All species within the genus share this generic name, but do not all have the same traits. Many closely related species to Clostridium botulinum are also highly toxigenic like the tetanus-causing bacteria Clostridium tetani and the gas gangrene causing Clostridium septicum. There are also non-toxigenic species like the soil microbe Clostridium sporogenes and gut microbe Clostridium butyricum [1].
There is variation within the species of Clostridium botulinum based on the specific neurotoxin that a particular strain produces. Botulinum neurotoxins, also known as BoNTs, are categorized A-G. For example, the strain C. botulinum 62A produces BoNT A1. In addition to different strains of Clostridium botulinum producing different BoNTs, the genes that encode for these toxins are located in different places in the bacterial genome. This leads to the recently discovered and perplexing inter-species exchange of bont genes.
Imagine if someone could give you a fragment of their DNA. Then imagine if this DNA codes for the ability to shoot poison out of your finger tips. Once you incorporate these genes into your genome, you gain this potentially advantageous ability. For Clostridium botulinum and many other bacteria, this is a reality. They can transfer and acquire genes from other bacteria, fragments of DNA, or viruses in their surrounding environment through a mechanism called horizontal gene transfer (HGT). Through this process, bacteria like C. botulinum might even be able to give or receive toxigenic genes. They have mobile elements of DNA or sections that can be easily copied and passed to a separate organism due to the complicated machinery housed in their cells [1].
One way that C. botulinum and many other bacteria add genetic material to their genome is through bacterial sex (conjugation). During this process the donor elongates a sex pilus that attaches to the recipient in order to facilitate close physical contact. A special mechanism called “Try machinery” bridges the small gap between donor and recipient allowing the transfer of DNA. Often the transferred genetic material is nestled safely on a donut shaped structure called a plasmid (Figure 3.) that is packaged separately from the bacterial chromosome. Like a package, plasmids are easy to send between bacteria, and this transfer can even take place between separate strains and species. Sometimes this process requires the help of a facilitator called a transposon. Transposons encode for molecules that cut and promote the movement of chunks of the bacterial chromosome or plasmids, making the transfer of genetic material even easier. Other times, no transposon is necessary and conjugation can happen autonomously [1].
Figure 3. Illustration representing bacterial conjugation of the bont gene harboring plasmid and the resulting toxigenicity represented by a lightning bolt symbol.
C. botulinum has several large plasmids that hold the majority of their toxin producing bont genes. In a paper by Nawrocki et al, researchers investigate if one specific plasmid, called pCLJ (Figure 3), requires the Tn916 transposon or if it can be passed simply through autonomous conjugation. They were specifically interested in plasmid pCLJ because it holds two bont genes. They set up experiments that allowed C. botulinum 657Ba, a carefully designed bacterial donor, to mate with several species of Clostridia and strains of C. botulinum. Some of the C. botulinum strains had bont genes and produced toxins while some of them were nontoxigenic. They did this to see if the bacterium could pass the pCLJ plasmid, bont genes and all, to a variety of other recipient bacteria. They analyzed the results and concluded that the pCLJ plasmid was passed to all recipient strains that did not already harbor a pCLJ plasmid.
Figure 4. An immunoblot that illustrates how the bont/a4 gene is associated with the pCLJ plasmid in donor and transconjugant strains, but is not present in recipients before conjugation takes place. P = the original parent strain of C. botulinum, D = the mutant donor, R = the pre-mating recipients, and T = post-mating recipients or transconjugants.
Researchers used an immunoblot to investigate if the donor bacteria transferred the associated bont genes to the recipient when plasmid pCLJ was passed through conjugation. This visual representation shown in Figure 4. illustrates that bont/a4, a bont gene present on plasmid pCLJ, was transferred and expressed. The original parent strain of C. botulinum is labeled as P while the mutant donor is marked as D. The pre-mating recipients are labeled as R, and post-mating recipients labeled as T for “transconjugant” (Figure 3 and 4). In the immunoblot, the bont/a4 gene is represented by the dark rectangles present in select columns. The row with bont/a4 genes is marked with an arrow and the characters pCLJ. It is clear that the bont/a4 gene has been passed to the transconjugant strains through conjugation based on the presence of dark rectangles in all of the T columns. Now that the researchers knew that the bont genes could be successfully passed to transconjugant strains, they were curious to see the consequences of these newly acquired genes.
With the knowledge that all of the originally nontoxigenic Clostridia strains acquired the pCLJ plasmid and the bont/a4 gene (Figure 4), they set out to see if these strains had the same killing capabilities towards vertebrates as the original C. botulinum parent strain. After injecting several groups of mice with different transconjugant strains, all mice in groups with the bont-harboring plasmid died, with the exception of sample 6 which was also incubated with an antitoxin. This shows that alongside accepting a bont gene, the originally non-toxigenic strains were given a newfound ability to produce murderous toxins (Table 1).
Table 3. Table indicating the 6 treatments used to inject groups of mice, 5 of which include the pCLJ bont-harboring plasmid. Sample 4 does not include the pCLJ plasmid and sample 6 was incubated with an antitoxin. The numerator in the “Day” columns represent the number of living mice on that date.
The results of the Nawrocki et al. study provide evidence that plasmids encoding for neurotoxins can be conjugately transferred from toxigenic Clostridium botulinum to non-toxigenic Clostridia. Consequently, mice injected with the once harmless bacteria were subject to death by botulism.
Akin to the way bacteria transfer beneficial genes in antibiotic resistance through conjugation, Clostridia coexisting in the intestines of vertebrates are evidently capable of spreading around a BoNT harboring plasmid through the same mechanism. Their coexistence in the stomachs of healthy dairy farm animals was already discovered in 2016, and presents a scary scenario for unknowing consumers of sub-optimally stored dairy products.
Outside of the agricultural industry, the research findings of Nawrocki et al. raises questions about the spread of BoNTs within Clostridia species in nature. Although their study reveals the frequency of BoNT plasmid transfer is relatively low, its potential to gain traction is not out of the realm of possibility. For non-toxigenic species, gaining a gene for a poisonous neurotoxin gives them a new advantage. If they kill the vertebrate whose intestines they were once peacefully living inside, they are creating a nutrient-rich decaying habitat to proliferate. The spread of this bacterial weapon could mean bad news for wildlife like the thousands of birds lost each year from avian botulism outbreaks. Nawrocki et al also bring up the phenomenon of BoNT transfer to already toxigenic species like Clostridium tetani or C. septicum. If these types of double-trouble scenarios were to gain prevalence in nature, the immune system of whomever is infected would be taking on an unforeseen challenge.
Figure 6. Photo illustration by TIME of a person getting several injections.
Lab-mediated bacterial mating to produce toxigenic strains gives medical research a new way to control this valuable poisonous substance. Studying and performing BoNT conjugation has the potential to aid the “production and purification” of botulinum neurotoxins used in up and coming medical treatments. In the last 20 years, the toxin has made its way into studies exploring its potential uses outside of cosmetics. Clinical trials are targeting different muscles with onabotulinumtoxinA injections to relieve migraines, muscle disorders, excessive sweating, and depression. A 2017 Time article explains these expanding therapeutic uses of the isolated botulinum neurotoxin. In addition to pain and muscle therapy, studying Clostridium botulinum conjugation could help us better understand how to combat botulism outbreaks in humans and wildlife in the unpredictable future.
Sources:
[1] Slonczewski, J., & Foster, J. W. (2016). Microbiology: An evolving science (Fourth ed.). New York: W. W. Norton & Company.
About the authors:
Rebecca Marsden ‘20 (right) and Juliet Greenwood ‘21 (left) met in their first biology class at Mount Holyoke College where they sat next to each other in the front row. Juliet is a Biology major with an Anthropology minor and a 5 College Certificate in Culture, Health and Science. As well as playing guitar and singing in the V8s, Juliet loves hiking and taking care of her many house plants. Rebecca is a Biology major with a Psychology minor, and is eager to learn more about microbial diversity and symbiotic relationships in future graduate studies. She spends her free time creating biological illustrations, working in her vegetable garden, and rock climbing.
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