Pharmacokinetics of botulinum neurotoxin serotypes: Implications for Infant Botulism

From MicrobeWiki, the student-edited microbiology resource

By Sarah R. Hall

Introduction


Clostridium botulinum is a rod-shaped, anaerobic, spore forming, gram-positive bacilli and is responsible for botulism (Slonczewski et al. 2009). In contrast to the genus Bacillus, the genus Clostridium does not show a highly similar DNA sequence, and the Clostridium species vary greatly throughout the Firmicutes phylogeny. The spores of Clostridium cells develop differently than that of Bacillus. The endospore swells at one end of the cell, which visually takes the shape of a “drumstick” (Slonczewski et al. 2009). The common habitat of Clostridium spores is the soil and water and will begin to germinate in anaerobic and substrate-rich environments (Slonczewski et al. 2009).


Electron micrograph of the Clostridium botulinum bacteria.http://www.kimicontrol.com/microorg/Clostridium%20botulinum.jpg

C. botulinum is able to produce seven different botulinum neurotoxins (BoNT/A to G) (Rossetto et al. 2006) that are responsible for four different types of human disease: wound botulism, food-borne botulism, adult intestinal colonization, and infant botulism (Fox et al. 2005). Only BoNT/A, B, E, and F cause disease in humans (Fox et al. 2005). There is an average of 110 cases of botulism reported in the U.S. per year, and infant botulism, first reported in 1976, is the most commonly reported botulism form contributing to about 70% of the reported cases. Infant boys and girls are affected equally with a mean age of onset of 13 weeks, with a range from 1-63 weeks (“Botulism: Epidemiological Overview for Clinicians”). The risk factors for infant botulism are living in a rural environment, having a parent who works in the soil industry, and the consumption of honey (Fox et al. 2005). In fact, 59% of reported cases in Europe are associated to honey. While only 20% of U.S. soil samples contain botulinum spores, most reports of infant botulism originate in California, Pennsylvania, and Utah (Fox et al. 2005).

Clostridial organisms infect, grow, and germinate within the gastrointestinal tract in infant botulism and adult intestinal colonization types and in the wound for the wound botulism type, and then begin to produce the toxins that are absorbed by the body (Fox et al. 2005). In food-borne botulism, on the other hand, the toxin is already formed, and ingestion of this preformed toxin is how the disease occurs.

The reason for the high incidence of infant botulism compared to the other three types of the human disease is that infants are much more susceptible to the colonization and growth of clostridia within the gut. Throughout the age window of 1-63 weeks, most infants are weaned from breast milk and introduced to much different foods. This change in nutrient type is paralleled with changes in the gut flora in the infant, and this altered gut flora presents an opportunistic environment for the clostridial spores to colonize the intestine (Fox et al. 2005). Animal studies support this view. It has been found that adult mice that were originally resistant to clostridial colonization became susceptible after administered antibiotics (a mixture of erythromycin and kanamycin sulfate), which changed the gut flora of the mice (Burr et al. 1982).

The clostridial spore germinates within the colon, and the BoNTs produced from C. botulinum travel from the intestinal lumen to the circulatory system, and finally to their specific targets, the peripheral cholinergic nerve terminals (Schechter 1999). In summary, BoNTs cause persistent, but reversible, inhibition of acetylcholine release and muscle paralysis. Inhibition of acetylcholine release occurs at the neuromuscular junction and autonomic nervous systems. Constipation is usually one of the first signs of a botulism infection in an infant (Fox et al. 2005). This is followed by a descending, symmetric motor weakness and paralysis. The autonomic nervous system functioning declines over the time period of hours to several days, affecting first the cranial nerves, then the trunk, extremities, and finally the diaphragm (Fox et al. 2005).

The diagnosis of infant botulism is usually electrophysiology, since it is the quickest method (Fox et al. 2005). Infant botulism is suspected when the amplitude of the M-wave is small, and when there is an incremental response to rapid repetitive stimulation. A longer test is laboratory identification of botulinum toxins or cells in the serum or stool(Fox et al. 2005). However, since constipation is usually a common side-effect, samples of the stool are more difficult to obtain. Further, serum testing has its own draw-back in that it is a less sensitive test in comparison to stool testing.

BoNTs are highly specific due to two reasons: First, the toxin is lethal at pico- to femtomolar concentrations (Rossetto et al. 2006). Particularly, in the very small body weight of infants, minuscule amounts of toxin (nanogram/kilogram body weight) can cause devastating consequences (Schechter 1999). Second, the ratio of the cholinergic presynaptic terminals to the entire exposed cell surface within the body is a very tiny ratio, making it evident that BoNTs are extremely specific (Rossetto et al. 2006). All seven serotypes of BoNTs are composed of two polypeptide chains: a light (L) chain of 50 kDa linked by one disulfide bond (Fox et al. 2005) to a heavy (H) chain of 100 kDa (Rossetto et al. 2006). The L-chain disulfide bond is linked to the first half of the H-chain (HN or N-terminal domain of the H-chain). HN is responsible for entry into the cell, while the HC domain (C-terminal domain of the H-chain) plays a role in the specific interaction of the neural binding (Rossetto et al. 2006).

The HC domain can be separated into the N-terminal half, which folds like the carbohydrate binding proteins of the legume lectin family, and the C-terminal half, which folds like the trypsin inhibitor family proteins (Rossetto et al. 2006). This bipartite structure of the HC has been presumed to account for the high affinity, specificity, and rapidity of neuronal binding. The HN domain is composed of two antiparallel α–helices, which are highly homologous among the BoNTs. This common structure in the HN domain among the BoNTs is translated into a common function, where the L-chain in all BoNTs undergoes transmembrane translocation at a low pH. The catalytic L-chain functions as a zinc-dependent endopeptidase (Fox et al. 2005) with a cleft shaped active site where a catalytic zinc atom is coordinated with one glutamate and two histidine residues (Rossetto et al. 2006).

Within the neuron, three proteins: VAMP (vesicle-associated membrane protein), SNAP-25, and syntaxin, all assemble to form a trimeric complex that is usually abbreviated as the SNARE complex (Rossetto et al. 2006). It is the collection of SNARE complexes in a rosette shape that facilitates the movement of the synaptic vesicle to the cytosolic side of the presynaptic membrane. Therefore, SNARE approximates the synaptic vesicle filled with neurotransmitter close to the neuron terminal membrane so that the vesicle membrane can fuse and discharge the neurotransmitter (in this case acetylcholine) into the synaptic cleft. Only the proteolysis of a single SNARE protein prevents the formation of a functional SNARE complex. BoNT/B, D, F, and G cleave only VAMP (VAMP and Sbr or synaptic vesicle protein are used interchangeably), where most of its cytosolic domain is lost. BoNT/A and E cleave SNAP-25 only within its C-terminus. BoNT/C cleaves SNAP-25 and syntaxin (Rossetto et al. 2006).

Section 1


Even though all botulinum serotypes result in a nonfunctional SNARE complex and subsequent prevention of the release of neurotransmitter, the duration of neuromuscular paralysis is very different among the BoNTs. Recent evidence has suggested that BoNTs/A, B, and C have a longer inhibitory effect on nerve terminals than BoNTs/E and F (Foran et al. 2003). Almost all of the infant botulism cases are caused by BoNTs/A and B. Type A is reported to occur at a higher frequency in the western half of the United States, while most cases of BoNT/B occur in the eastern half the of the United States (Fox et al. 2005). Foran et al. 2003 used rat cerebellar neurons in order to quantify the half-life of each BoNT’s effect and the rate at which the SNARE substrates were made functional again. The cerebellar neurons were bathed in a culture medium with certain concentrations of BoNT/A (Figure 1A; 0, 0.1, 10 pM) or BoNT/B (Figure 1C; 0, 10, 100, 1000 pM) for 24 hours. In order to measure neurotransmitter release, the amounts of Ca2+-dependent [14C]glutamate released into basal and K+-stimulated samples was calculated and expressed as a percentage of the total cell content (Figures 1A and C). Further, in order to quantify the relative speed at which the SNARE substrates were made functional again in response to increasing concentrations of BoNT/A (Figure 1B) and BoNT/B (Figure 1D), equal amounts of neuronal protein were immunoblotted using SMI-81 Ig (SNAP-25) and anti-HV62 (for Sbr2) in order to measure the degree of SNARE proteolysis.

Interestingly, it was found that BoNT/A showed a concentration-dependent inhibition of neurotransmitter release to about a maximum of 65% blockade (Figure 1A) and corresponded to the cleavage of approximately 90% of the SNAP-25 (Figure 1B). BoNT/B, on the other hand, induced a dose-dependent inhibition of neurotransmitter release of about 80% blockade (Figure 1C) and was associated with proteolysis of about 100% of Sbr2 (Figure 1D). Even though BoNT/B showed a much greater inhibition of neurotransmitter release and more extensive proteolysis of the substrate, the removal of BoNT/A and BoNT/B by washing, suggested that BoNT/B shows a shorter duration of inhibition than BoNT/A. The disinhibition of neurotransmitter release and replenishment of the Sbr2 substrate for BoNT/B occurred in a time-dependent fashion for the neurons at 2, 7, 25, and 28 days. The time-dependent recovery of release and SNAP-25 replacement for BoNT/A was not seen (Figures A and B).

Follow-up experiments confirmed a lack of any significant recovery of neurotransmitter release a month after BoNT/A incubation, whereas BoNT/B had a much faster recovery period. Exponential decay calculations obtained inhibitory half-lives (t1/2 INH) for BoNT/A exceeding 31 days, while the mean t1/2 INH for BoNT/B was approximately 9.84 days (Table 1). The same concentration dependency studies were used to calculate the t1/2 INH for BoNT/C, E, F, and tetanus toxin (TeTx) as shown in Table 1. Therefore, BoNT/A is the longest-lasting, while BoNT/E and F are very short lasting and BoNT/B can be considered intermediate in duration.

Section 2


Include some current research in each topic, with at least one figure showing data.

Section 3


Include some current research in each topic, with at least one figure showing data.

Conclusion


Overall paper length should be 3,000 words, with at least 3 figures.

References

“Botulism: Epidemiological Overview for Clinicians.” Emergency Preparedness and Response. 2006. Centers for Disease Control and Prevention.

Burr, D. and Sugiyama, H. “Susceptibility to enteric botulinum colonization of antibiotic-treated adult mice.” Infection and Immunity. 1982. Volume 36. 103-106.

Fox, C., Keet, C., and Strober, J. “Recent Advances in Infant Botulism.” Pediatric Neurology. 2005. Volume 32. p. 149-154.

Rossetto, O., Morbiato, L., Caccin, P., Rigoni, M., Montecucco, C. “Presynaptic enzymatic neurotoxins.” Journal of Neurochemistry. 2006. Volume 97. 1534-1545.

Schechter, R. “Infant Botulism: A Brief Overview.” Anaerobe. 1999. Volume 5. 161-164.

Slonczewski, J. and Foster, J. Microbiology: An Evolving Science. New York: W.W. Norton & Company, 2009.




[Sample reference] Takai, K., Sugai, A., Itoh, T., and Horikoshi, K. "Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney". International Journal of Systematic and Evolutionary Microbiology. 2000. Volume 50. p. 489-500.

Edited by student of Joan Slonczewski for BIOL 238 Microbiology, 2009, Kenyon College.