Question

Your roommate Tina bravely eats that leftover potato salad that had been stored in the fridge for far too long and now she is nauseous.  She asks you, the Conscientious Biology Student, what has happened regarding her biology.  What is the pathway for how toxins from bacteria (within bad potato salad) can ultimately lead to a vomiting response?  What structures and neural pathways are involved?

Answer 1

After consumption of the stale potato salad, she has been infected with Clostridium botulinum endotoxin. Traditionally, foodborne botulism has been associated with underprocessed and abused sausages or home-canned foods; however, in recent years botulism has been acquired through the consumption of contaminated foods such as potato salad, sauteed onions, garlic sauce, cheese, yogurt, bean paste, and olives.

Clostridium spp. are spore-forming bacteria, members of the family Bacillaceae and include obligately anaerobic or aerotolerant, sporeforming rods that do not form spores in the presence of air and, at least in early stages of growth, are usually Gram-positive. The endospores of many species are extremely sturdy and survive extended boiling in water and exposure to air. Spores germinate under conditions favorable for vegetative growth, such as anaerobiosis and presence of organic substrates. Cl. botulinum are motile by means of peritrichous flagella and produce botulinum neurotoxins, the most lethal poison known. There are seven types of botulinum neurotoxin, A through G, based on the antigenic specificity of the toxin produced by each strain.

Symptoms of botulinum neurotoxin ingestion appear 12–36 h after consumption of contaminated food and initially may include nausea and vomiting. However, these symptoms are followed by the more characteristic neurological signs including visual impairment and acute flaccid paralysis that begins with the muscles of the face, head, and pharynx, descending to involve muscles of the thorax and extremities and leading to possible death from respiratory failure caused by upper airway or diaphragm paralysis. The minimum toxic dose of Cl. botulinum neurotoxin has not been determined, but from a human health and food safety standpoint, there should be no tolerance either for the neurotoxin itself or for conditions allowing the growth of the organism in foods.

Botulinum neurotoxin is synthesized during cellular growth and is subsequently released during cell lysis, where proteolytic cleavage activates the molecule. There are four categories of botulism, which include the classic foodborne botulism derived from the ingestion of preformed toxin in foods, wound botulism resulting from toxin production after organism growth in an infected wound, infant botulism from toxin elaboration in the intestinal tract of infants, and botulism due to intestinal colonization in older children and adults with intestinal disorders or complications resulting in a lack of microbial competition. Botulinum neurotoxin introduced in any of these categories is transported via the bloodstream to neuromuscular junctions, where the toxin irreversibly binds to receptors on peripheral nerve endings and subsequently is internalized into the nerve cell.

Structure of botulinum toxin

There is currently little known about the genes that encode for the neurotoxins and the assembly and production of the molecule. Numerous studies have yielded inconsistent results, but the structure of the neurotoxin has in fact been discovered. The toxin is in the form of a noncovalently bound complex that contains several nontoxic proteins that consist of hemegglutinin and nonhemegglutinin, and weighs 150 kDa (Sebaihia et al. 2007). In order for this polypeptide molecule to become toxic, it is cleaved by a protease at one-third the distance from the N terminus. The exact enzyme that performs this function has still yet to be been determined. This action yields two fragments: a smaller, lighter fragment weighing 50 kDa and a heavier fragment with a larger weight of 100 kDa. These two fragments are kept joined together by a disulfide bond and collaborate to produce the damaging biological response (Todar 2009).

This is the structure of botulinum toxin

Neural pathway involved

The neurotoxins target the body’s peripheral nervous system, which is greatly exposed to pathogens, as it is not protected by the blood-brain barrier or bone. They pass through the membrane and enter into the neuron cell by endocytosis. Pirazzini et al. hypothesized that the heavier chain forms a channel within the membrane in which the light chain can then pass through into the cytoplasm (Pirazzini et al. 2013). They found evidence that the heavier chain contains two polysialoganglioside binding sites that allow the toxin to successfully bind to the membrane and use the low pH environment on the outside of the cell to drive its entry into the neuron which has a neutral concentration. The study also discovered that 37 degrees Celsius is the optimum temperature for the transport of the toxin through the plasma membrane, as the translocation of the lighter chain into the cell occurred in just minutes, which is an extremely rapid pace.

Image taken from - https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618376/

Once inside the neuron, the toxin binds to the presynaptic membrane of the cholinergic nerve terminals, blocking the release of the neurotransmitter acetylcholine. Acetylcholine plays an essential role in the body, as it is responsible for regulating the somatic nervous system, which controls the voluntary movements of the skeletal muscles, and is the only of its kind. No other neurotransmitter initiates this type of movement. As a result, if the toxin does in fact bind to the receptor it has very damaging effect, as this blocking mechanism prevents the nervous system from communicating with the muscles, resulting in limited muscle movement and paralysis.

The botulinum toxin produces specified cleaving proteases that allow the pathogen to successfully attach to the synaptic vesicles. Studies have identified the synapse as the synaptic vesicle protein SV2 (Peng et al. 2010). If this specific receptor SV2 is not present within a cell then the neurotoxin does not produce the same effect. Polysialogangliosides and SV proteins surround the membrane to facilitate the binding of the neurotoxin.

The carboxy-terminal domain of the heavy chain recognizes a specific binding site, while the nitrogen-terminus transports the lighter chain into the nerve cytosol (Peck et al. 2010). The lighter chain contains metalloproteases that target specific proteins involved in controlling the exocytosis machinery (Verderio et al. 2006). The inhibition of this integral machinery stops the release of acetylcholine and the neuron fails to send an important signal throughout the body. The lighter chain also decreases the stability of the binding complex, further preventing acetylcholine from being able to bind to the synaptic vesicles (Peck et al. 2010).

Image taken from - https://www.asmalldoseoftoxicology.org/toxipedia/

In addition to releasing neurotoxins when exposed to varying environmental conditions, Clostridium botulinum also increases production of proteases that are secreted from the cell to breakdown polypeptides to contribute to contaminating food and therefore increasing its own toxicity. A large proportion of the bacteria’s genome encodes for several different variations of protease enzymes (Sebaihia et al. 2007).

References:-

1) https://www.nature.com/articles/srep13397

2) https://microbewiki.kenyon.edu/index.php/Clostridium_botulinum_Neurotoxins

3) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604998/#b7