Bacteria Producing Neurotoxin Responsible For Botulism In Humans Biology Essay

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Clostridium botulinum G is a rod shaped gram positive bacteria and an obligate anaerobe which was recently reclassified as Clostridium argentinense. It produces neurotoxin G (as a single polypeptide chain) which causes botulism in humans. Botulism is characterized by paralysis caused by the inhibition of acetylcholine release which may result in respiratory failure.

Neurotoxin G acts on the neurons of the nervous system as do the other botulinum toxins (A to F). They enter the neurons and prevent the release of acetylcholine and therefore nerve signal transmission. At the neuromuscular junctions this causes paralysis, which is the main symptom. The mechanism of action of all of the neurotoxins is similar with the difference lying in where the toxin cleaves the protein responsible for the release of the acetylcholine. Botulism can be classified as Foodborne botulism, Wound botulism, Infant botulism, Adult botulism and inadvertent botulism. Depending on how the bacterium infects the host (route of transmission) or who the host is.

Neurotoxin G has been found to bind to the SNARE (soluble N-ethymaleimide-sensitive factor attachment protein receptor) proteins within the cell. SNARE proteins play an indispensible role in the release of acetylcholine by allowing the fusion of the vesicles containing acetylcholine to the pre-synaptic membrane which occurs in the presence of calcium (Ca2+). This causes the release of the acetylcholine into the synaptic cleft, and therefore the transmission of the nerve impulse. Neurotoxin G is endocytosed in a vesicle into the neuron after it binds to its membrane. The neurotoxin has an N-terminal translocation domain which forms part of the heavy chain which undergoes a conformational change inside the endocytosed vesicle due the low pH. This change allows the movement of the light chain into the cytosol of the neuron. Once inside the neuron the light chain has a high affinity for the SNARE proteins, where it cleaves a particular snare protein called synaptobrevin and prevents the mechanism described above and therefore stops transmission of nerve impulses and causes paralysis. 6

The bacteria colonises the intestinal tract from which the toxins are absorbed and transported to neuromuscular junctions where it causes paralysis. The initial food borne botulism symptoms are nausea, vomiting, diarrhoea and adnominal cramps. After the toxins are transported to the neuromuscular junctions the neurological symptoms begin with dry mouth and blurred vision followed by the characteristic symmetrical descending flaccid paralysis. In the case of wound botulism the progression of the disease is similar but with a later onset of symptoms but no gastrointestinal symptoms. Adult botulism occurs in adults who have compromised normal flora due to surgery of the use of antimicrobials. Normal flora provides protection against infection by providing competition for the bacteria. Infant botulism causes similar symptoms to foodborne botulism and adult botulism. The first indication of infant botulism is constipation and after a longer period of time poor feeding becomes evident followed by progressive weakness which marks the beginning of the neurological symptoms.

When botulism is suspected the most effective method of confirming the diagnosis of botulism is by performing a mouse bioassay. This involves the injection of serum or the fluid extracts of faeces from the patient into the peritoneum of the mouse. If the mouse then shows symptoms associated with botulism then it is safe to assume the patient has botulism. However, to correctly identify the type of neurotoxin causing the botulism a further mouse bioassay has to be carried out. Each mouse is given an antitoxin for a specific neurotoxin. Then they are injected with the patient’s serum or faeces fluid extracts. The mouse that has the correct antitoxin will survive therefore allowing the identification of the neurotoxin causing the botulism. Biochemical tests may be carried out to confirm the general diagnosis of botulism which include a negative catalase test or a negative spot indole test. 3 4

Enzyme-linked immunosorbent assay (ELISA) is a molecular technique used to detect the presence of the neurotoxin in the patients stool samples. This technique involves the use of rabbit and goat antibodies for the neurotoxin. Rabbit anti-type G IgG is fixed onto a plate to which the patients sample is added followed by goat anti-type G IgG. The neurotoxin is sandwiched between the goat and the rabbit anti-type G IgG. This is then followed by the addition of a further rabbit antibody which is specific for the goat anti-type G IgG. This rabbit anti-type G IgG is conjugated with alkaline phosphatise so that when an enzyme substrate is added it will produce a coloured product. If no neurotoxin is present then no coloured product would appear. However, if the neurotoxin is present a coloured product would be present. This technique is less reliable than the mouse bioassay but it is cheaper and faster. 1

Clostridium argentinense is the least common of the clostridium botulinum species which causes botulism. With only a few cases being confirmed since the bacteria was first isolated in Argentinean soil. The fact that the virulent factor of the bacteria is the secreted neurotoxin means that a relatively small amount of bacteria is needed to cause the disease. The bacteria forms spores in unfavourable conditions and germinates in favourable ones, allowing it to survive in harsh conditions increasing its ability to spread. It is then that it begins to produce large amounts of the neurotoxin. Susceptibility for Clostridium argentinense or other forms of clostridium botulinum is not affected by age, sex or race. However, infection is more likely to infect and cause disease in immunocompromised hosts, whose normal flora is damaged. Furthermore, there are various methods of infection such as through contaminated foods or wounds.

Clostridium argentinense has only been reported a few times to cause botulism. However, as mentioned a small amount of bacteria produce large amounts of the toxin, making mortalities more likely even with a relatively small infection. In general the mortality rate for botulism today is 15% which is markedly lower than in 1900. This may be due to better protections for food, however, because the bacteria sporulates food borne botulism is still common. The incidence rate in the United States is high with 724 cases of food born botulism. However, botulism caused by Clostridium argentinense which resulted in death was only recorded in 5 corpses after an autopsy. Treatment for botulism is usually amined at countering the toxins and so antitoxins often derived from horses are given. 5 2

In summary botulism is a prevalent threat, especially in under developed areas of the world where treatment is not always available and preventative measures are not usually taken. However, botulism caused by Clostridium argentinense is still understudied and not fully understood.

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