Sometimes, when your mom is pestering you to do something, she has a point! You just enjoyed a delicious meal and want to save it for later, if she says put it in the fridge, you must listen. Why? Because of a life-threatening illness named botulism.
Botulism sounds like some crazy disease that was around in the middle ages, but this crazy disease is actually very present in this era now! It is caused by a nerve toxin produced by the bacterium Clostridium botulinum. C. botulinum is a gram-positive, rod shaped, spore forming, anaerobic bacteria, and is often linked to improper food refrigeration, canning, and contamination. Botulism is at times a fatal disease, with symptoms being muscle weakness, double vision, and breathing issues, often taking months for an individual to recover. Interestingly enough, C. botulinum itself does not always cause botulism, meaning it can be present in your food, and not get you sick.
Sounds confusing though right? How does C. botulinum cause botulism sometimes and not other times? Does it pick and choose? Does it have a checklist or a vendetta against certain foods?
Well, it all starts with sporulation. Sporulation is the process of forming spores, which is a survival method in bacteria specifically to withstand harsh conditions. In this instance, C. botulinum undergoes environmental stress like starvation, lack of oxygen, or improper temperature conditions, and decides to produce spores that are highly resilient. To further illustrate, if you left your favorite homemade salsa out on your kitchen counter, and C. botulinum is present within it and doesn’t find the warm temperature favorable, expect some spores in your favorite salsa.
During sporulation, C. botulinum produces a potent toxin, the botulinum neurotoxin (BoNT). This neurotoxin blocks neurotransmission causing paralysis. Interestingly enough, BoNT production is not always made. In fact, more commonly, C. botulinum produces harmless spores, which can be ingested and not cause illness. In fact, the bacteria exists prevalently in the environment, often being found in soil, sediment, and dust. However, BoNT specifically grows in low-oxygen and low-acid environments (i.e. food being improperly canned). Evidently, toxin expression for C. botulinum has been shown to occur during growth periods and sporulation.
Life cycle of C. botulinum sporulation pictured. Image credit to Lina Korkiakoski (Metropolia University of Applied Sciences).
This paper written in 2023 by Mertaoja and others from the University of Nottingham dives into more detail on this matter. It explains that toxin production and sporulation are the main reasons for pathogenesis in C. botulinum. The spores allow for long-term survival of these toxins that later lead to infection. Research also shows that in certain C. botulinum strains, low temperature activates neurotoxin promoter in the spores, induces twin sporulation, and co-activates sporulation and toxin production in the population (and this is why a fridge comes in handy). Therefore, this research analyzes the effect of low temperature in sporulation with C. botulinum.
Conditions of the environment have been shown to greatly affect the ratio of toxinogenesis and sporulation for C. botulinum. At 30 degree celsius, cultures were undergoing rapid cell death, while at 10 degrees celsius, the population of cells were thriving better and balanced. C. botulinum spores thrive in nature even through harsh conditions, meaning they can be around for a long time.
We might be slightly confused at this point… We know that sporulation causes BoNT, but what is responsible for the mechanism of sporulation? Who is the evil genius that keeps toxin production and sporulation active when C. botulinum is stressed out?! Well, the mastermind behind all these mechanisms is Spo0A.
Spo0A is the transcriptional regulator that controls the initiation sporulation. In other words, the master switch that starts sporulation. Transcriptional regulators are proteins that control expression within a cell by binding to specific DNA promoters or enhancers. In C. botulinum, Spo0A has been shown to positively regulate BoNT production by binding to bontE promoters. This evidence suggests that toxin production and sporulation are activated by Spo0A-driven cellular programs.
Data collected from this study shows that sporulating cells release BoNT through mother-cell lysis when a mature spore is released. Cell lysis is essentially a process where the cell ruptures, releasing all internal contents. In C. botulinum, mother-cell lysis is the death of a mother cell once a new mature cell is formed, this sudden rupture of the mother cell is what releases the spore contents. In this study, the mutant Δspo0A was used to contrast Spo0A as a regulator. The mutant was bound to a BoNT gene promoter, and these cells were shown to produce no spores during mother-cell lysis, and it reduced BoNT levels down to 10%. This gives more reason to believe that cells release BoNT through Spo0A mediated autolysis.
C. botulinum culture pictured. Image credit to Wikipedia.
C. botulinum type E cultures were used throughout this study, these cultures are also known as the wildtype (WT) since the strain naturally occurs in nature within marine freshwater sediments, fish, and other aquatic animals. The study suggests that up to 97% of the neurotoxin was released in WT cultures, while the Δspo0A cultures only released 2.5% of it. Once again, suggesting a correlation between spo0A in cell lysis and sporulation. Δspo0A also maintained their shape in comparison to WT cells (WT cells were more sporulated).
The study also examined BoNT gene expression and sporulation in C. botulinum populations by changing the temperature of the populations. The bacteria was grown on a cooked meat medium-TPGY plate, as well as a CMM-TPGY plate at 30°C for 96 h or at 10°C for 60 days. SNAPCd, which is a system that monitors protein production, gene expression during sporulation and growth (SPO+ or SPO-), and toxin gene promoter activation (TOX+ or TOX-), was analyzed using fluorescence microscopy using membrane dye Mitotracker Green (MTG) on the cells.
Figure 2 of the study “Cellular and population strategies underpinning neurotoxin production and sporulation in Clostridium botulinum type E cultures”.
In figure 2a above, the location of the TMR-Star SNAP signal (red) was analyzed for sporulating cells that were recently engulfed (phase-dark) and matured (phase-bright) forespores. the SNAP signal in the cells with the phase-dark forespores (under phase contrast) was found in the mother cell, forespores, or both. In cells with the phase-bright forespores, the SNAP signal was found only in the mother cell. To add, Figure 2b shows the population structure at different times concerning the production of the toxin and sporulation. In addition to this, the growth of the cells was measured using optical density at 600 nm. Finally, figure 2c, shows the neurotoxin concentration in this image as the light gray bars, and culture supernatants as the dark gray bars.
That sounds complicated, so let’s break that down. Transcription from the neurotoxin gene promoters were shown during vegetative growth (SPO-) and sporulation (SPO+), meaning that this bacteria actively transcribes and expresses these toxin genes during both of the main life cycle stages. In addition, the production of the BoNT toxin was reported in temperatures as low as 3°C. At 30°C, TOX and SPO were happening at different times and only overlapped during the transition phase, and by day 4 the population mainly had spores and lysed cells. However, at 10°C TOX+ cells appeared later, and more than half of them appeared after 1 month, yet even by month 2 the culture consisted of mainly vegetative cells. This suggests that this C. botulinum toxin is more stable and active in cold temperatures. All in all, the results show that in C. botulinum Type E strains, low temperature supports long-lived and diverse populations, activates neurotoxin promoters in the forespore, and co-activates sporulation and toxin production in the population.
It’s important to note that this study examined a strain of C. botulinum that often releases toxins in temperatures as low as 3°C, which is one of the reasons improperly vacuum packaged foods that are refrigerated can cause botulism. To exemplify, we have seen botulism cases before with seafood products. However, there are strains of C. botulinum that thrive in warm environments. For this reason, it is important to research how to properly store your food, to avoid contamination and risky conditions. Unfortunately, there have been many cases where improperly canned foods at warm temperatures or even room temperature have caused botulism.
Evidently, there are many mechanisms playing a role in the infection we know as botulism. This research study by Mertaoja and others found significant proof of BoNT production and the role it plays during sporulation. These findings can help scientists and companies get to the bottom of this disease and figure out detailed ways food items should be properly stored to avoid sporulation in C. botulinum. For specific directions, research on bacterial toxin production and sporulation can be implicated further by analyzing gene expression and traits in single cell levels.
Clearly, getting infected with this bacteria can be dangerous and at times deadly. So, let’s make sure to listen to our mom just this one time, and put your leftovers in the fridge!
About the Author:
Yeslee Neris '26 is a graduating senior at Mount Holyoke College majoring in Biological Sciences and Latin American Studies. After graduation, Yeslee will be pursuing her Masters in Nursing at Johns Hopkins University! Her favorite part of taking Microbiology at Mount Holyoke was learning about the different functions and mechanisms between microbes, specifically, she found virulence fascinating.
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