Clostridium botulinum Neurotoxins
Introduction
Clostridium botulinum is a gram-positive, rod-shaped bacterium and pathogen that is prevalent in marine and soil environments around the world. As obligate anaerobes, Clostridium botulinum must live in low oxygen habitats, as higher concentrations are toxic to the cells. These bacteria also live in relatively neutral environments and have the most successful growth rates in a pH ranging from 4.6-7.0. Clostridium botulinum is most commonly found as an inactive spore in the shape of an oval. The spores generate a tough outer protective coating and several layers of membranes to enclose the cell and keep it alive. Most Clostridium botulinum spores reside on the surfaces of fruits, dairy products, vegetables, seafood, and various other canned foods. As spores, the bacteria usually remain relatively harmless, but when they are activated and resume growth, the bacteria release several different types of potent neurotoxins that can cause the paralyzing disease botulism. The production of the neurotoxins acts as a defense mechanism for the bacteria to protect them from intense heat, increased acidity, and possible fragmentation and damages. Clostridium botulinum can produce up to seven different types of toxins ranging from A-G. While all forms of the toxin are destructive, types A, B, E, and F are known to specifically cause botulism in humans, while types C and D are associated with animal botulism. There are several different mechanisms in which this disease can be transmitted, but Clostridium botulinum most usually infects individuals by contaminating canned or unrefrigerated food, infecting a wound, or entering a key water source.
Pathology
The discovery and detection of new emerging neurotoxins found in contaminated food has fueled a growing concern for ensuring that safe food is distributed in numerous countries around the world. Many recent studies have investigated new techniques in which to regulate Clostridium botulinum growth and diversification. This requires improved mechanisms of detection of this bacteria and a comprehensive understanding of how and why it flourishes in the environments that it does and how exactly it causes such a biological response. While reported cases of botulism around the world are relatively infrequent, these bacteria are still a hazard. The smallest microscopic amounts of the neurotoxins released by these bacteria can cause the most damaging form of the disease. It has been reported that only 30 ng of the neurotoxin can induce a drastic effect (4). An antitoxin can be administered to help reduce the effects of the disease, as it inhibits the neurotoxins that have yet to bind to the nerve receptors decreasing the toxin’s ability to cease muscle movement. Infants are most susceptible to infection due to their underdeveloped intestinal microflora, allowing for greater colonization of the bacteria (7). Clostridium botulinum types A and B mainly cause infant botulism, but types C, E, F, and G have also been identified as causative agents.
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 (4). 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 (6).
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 lighter chain can then pass through into the cytoplasm (9). 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 with a neutral concentration. This study also discovered that 37 degrees Celsius is the optimum temperature for the transport of the toxin through the plasma membrane. When the neurons are in 37 degrees Celsius, the translocation of the lighter chain into the cell occurred in just minutes, which is an extremely rapid pace. When the Pirazzini et al. tested the effects of 20 degrees Celsius on toxin transport they found that no toxins entered the neuron. These results indicate that transfer of the toxins through the membrane is greatly dependent on temperature.
Once inside the neuron, the toxin binds to the presynaptic membrane of the cholinergic nerve terminals, which are responsible for releasing the neurotransmitter acetylcholine. 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 (11). 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 lighter chain contains metalloproteases that target specific proteins involved in controlling the exocytosis machinery (5). Without this integral machinery, the release of acetylcholine is inhibited and the neuron fails to send an important signal throughout the body. 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.
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 help contaminate food and increase its own toxicity. As a result, a large proportion of the bacteria’s genome encodes for several different variations of protease enzymes (4).
Forms of Prevention
High-pressure thermal treatments are most commonly used as a remedy to remove all of the harmful bacteria, even when they are still in the form of a spore. If the spores are treated with heat of 100 degrees Celsius for over an hour, the spores are inactivated and unable to produce toxins (9). Cooking has become an easy prevention technique to ensure safe preparation of food. While food corporations have used this method for many years, new cases where certain food products or sources are contaminated continue to arise, and novel mechanisms for regulation must be tested.
Genome Structure
This species can be categorized into four genetically and physiologically distinct groups that all produce different types of neurotoxins. The Clostridium botulinum in group I produce the toxin types A, B, and F. They are proteolytic and have an optimal growth temperature of 37 degrees Celsius. This group of toxins most commonly causes infant botulism and contaminates food. Group II consists of Clostridium botulinum that produces type B, E, and F toxins and is mostly found in contaminated food products. They are nonproteolytic and develop at a lower optimal temperature of 30 degrees Celsius. Group III contains toxins type C and D and are most commonly associated with botulism in animals. Group IV produces toxin type G. The genome for the group I bacteria has been fully sequenced revealing that this bacteria contains DNA and a plasmid. The chromosomal DNA has 3,886,916 base pairs, which carries 3,650 genes, and the plasmid is composed of 16,344 base pairs, which carries encodes for 19 genes (4). A large proportion of the genome codes for proteases that are used for the metabolism of proteins. The sequencing of this genome revealed that there has been no recent integration of new foreign DNA. This suggests that this species is incredibly stable and lives in a nonthreatening environment, as no new genes were acquired for increasing protection, enhancing metabolism, or increasing movement to or detection towards food sources (4).
Cell Structure
Clostridium botulinum is a spore-forming, gram-positive firmicute. These bacteria have the ability to form spores even when in a thriving environment. The formation of spores does not serve the sole purpose of protecting and storing the cell, but it provides the means for the bacteria to release neurotoxins when they begin germinating. The factors to initiate germination greatly vary and depend on where the spore is located (1). In many food products, spores are activated by salt and pH concentrations, or by the addition of chilled or heated temperatures for either storage or preparation. There is usually a small concentration of spores present in a given food sample, but it only takes a very scarce amount to infect an individual. Through designed experiments, a model could be generated to predict the growth rate of the bacterium and determine what conditions initiate the activation of a spore (1). This could lead to the discovery of improved prevention techniques to ensure that food is safe and free of Clostridium botulinum. A study by Webb et al. demonstrated that the rate and amount to which spores germinate is directly related to the proximity of spores to each other. The higher the density of spores in the same location, the more overall germination occurred (1).
Metabolism
Clostridium botulinum can fully metabolize amino acids and chitin, and can partially metabolize several other polysaccharides. Proteases secreted by the cell can cleave surrounding polypeptides so the bacterium can digest smaller molecules (4). There is evidence in the genome that this bacteria uses fermentation pathways that utilize a series of coupled oxidation-reduction reactions to acquire energy, where the oxidation of one amino acid is directly coupled to the reduction of another amino acid. Studies have compiled evidence that the amino acid glycine is first reduced by a glycine reductase complex and then oxidized by a glycine cleavage system (4). It has been shown that this bacteria ferments glycine, proline, phenylaline, and leucine. The energy from fermenting amino acids provides the species with a significant amount of energy. The remaining energy is obtained by sugar metabolism. Clostridium botulinum uses the second most abundant sugar, Chitin, as its main source of polysaccharide energy (4). Chitin is commonly found in the exoskeletons of arthropods and insects, such as lobsters and mollusks, and in the cell walls of fungi, both of which are prevalent in the marine and soil environments in which this bacteria resides. Clostridium botulinum’s genome encodes five different enzymes that are capable of metabolizing this sugar. In addition to being a source of energy, chitin can also provide supplementary carbon and nitrogen. Starch is a potential source of energy, but these bacteria do not have all of the enzymes required to fully degrade this polysaccharide. Several types of Clostridium botulinum have a complete glycolysis system in addition to having the capability of fermentation (4).
Infant botulism
Infant botulism appears in infants less than one year old, and is caused due to the lack of microflora in an infant’s intestines. The lack of developed microbial communities can leave a newborn unprotected when encountering foreign bacteria ingested from unknown foods. Without the complete microflora, the Clostridium botulinum spores are able to germinate, which releases the threatening neurotoxins. Type A and B toxins are most commonly released, but types C, G, F and E have also been witnessed to cause infant botulism (7). The most commonly contaminated food that carries infant botulism is honey. The number of Clostridium botulinum spores have been calculated in samples of honey and have been measured to range from 1 to 60 spores. Even a small number of less than ten spores is still more than enough to produce an overwhelming amount of neurotoxins (7). The spores are only activated when they come in contact with the environment of the intestines. The conditions created by honey keep the spores in an inactive state. Honey maintains a relatively anaerobic environment due to the viscosity of the substance and retains a low pH. It also has an extremely high sugar concentration and a low protein concentration. Clostridium botulinum relies on amino acids as an essential nutrient for energy, and could possibly form spores in response to a lack of substrate. It could also form spores due to the anaerobic environment. This can be viewed as beneficial, as it does not allow for the release of harmful toxins in a prevalent food, but it could also be seen as potentially damaging. If these bacteria remain in the form of a spore due to environmental conditions, this suggests that they could be present in honey samples for essentially as long as they can retain their spore from (7). Bacteria use sporulation as a mechanism in which to survive for as long as needed until a favorable environment is reached. This suggests that theoretically Clostridium botulinum could potentially survive and persist in honey samples for years undetected.
The results of an experiment conducted by Nevas et al. testing the concentration and type of Clostridium botulinum in honey samples from different northern European countries concluded that toxin types B and A were most prevalent in the Danish and Norwegian honey samples, while only type E neurotoxin was found in the Swedish honey samples (7). Overall, there was more bacterium present in the Danish honey than in other of the other samples measured from surrounding countries. Nevas et al. hypothesized that the Clostridium botulinum spores could have originated from a nearby water source that came in contact with the honey samples. This would suggest that concentration of bacterial spores in honey reflects the total concentration of bacteria present in the natural environment, which would imply that the soil and aquatic environments in Denmark contain the most of these harmful bacteria.
Food-borne botulism
Smelt et al. conducted an experiment that attempted to discover or gain better insight into how non proteolytic Clostridium botulinum can grow and develop at refrigerated temperatures (8). This experiment would be especially beneficial for keeping dairy products safe from the bacteria’s neurotoxin. It had been previously determined that heated spores display a much different development process than unheated spores. The results of this experiment showed that there were less spores present in the colder samples, but this result was not due to the cold temperatures. The bacteria in the colder temperatures had growth and replication rates that were in normal ranges. The colder temperature could have influenced other conditions, such as oxygen levels or type of media that then influenced the prevalence of the Clostridium botulinum (8). 90% of the spores treated with a heated condition could not germinate, and a considerable proportion could not replicate. While this study was not able to generate any conclusions as to why there are smaller concentrations of these bacteria in refrigerated foods but no change in growth rate, it did demonstrate that Clostridium botulinum could survive in colder conditions. This could provide possible implications for refrigerating foods that potentially contain this bacterium.
Conclusion
While there are currently successful methods to rid of Clostridium botulinum from contaminated food sources, further research is still in need to ensure that this bacteria presents no threat, as botulism is one of the most devastating diseases. By gaining a more comprehensive understanding of the structure of the Clostridium botulinum neurotoxins and how they function in the body, new ideas can be generated as to how to cure this disease and prevent it from even occurring.
References
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