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The Dirt Is Mightier Than The Sword Tetanus Toxin in the Human Body Whitefish Bay High School SMART Team Cara Ahrenhoerster, Quinn Beightol, Gabe Drury, Xavier Durawa, Riley Gaserowski, Shirley Hu, Domi Lauko, Ciara Otto, Andrew Phillips, and Helen Wauck Teachers: Mike Krack and Paula Krukar Mentor: Joseph Barbieri, Ph.D. Medical College of Wisconsin Abstract Introduction Tetanus neurotoxin (TeNT), one of the most potent toxins for humans, causes paralytic death to thousands of humans annually. TeNT is produced by the bacterium, Clostridium tetani, an anaerobic bacterium usually found as spores in soil. C. tetani often infects humans through open wounds where the bacterium colonizes the infected tissues. There are two domains of TeNT: the A domain possesses catalytic activity, while the B domain is made up of two subdomains: the translocation sub-domain and receptor-binding sub-domain. SNARE protein, VAMP-2, is the target of the catalytic A domain of the TeNT. This SNARE protein regulates fusion of synaptic vesicles with the plasma membrane of the neuron, allowing the release of neurotransmitters that are responsible for relaying nerve signals such as inhibitory impulses to the body’s muscle cells. TeNT binds to dual receptors, two gangliosides, on the presynaptic membrane of α-motor neurons. TeNT hijacks the trafficking machinery of the motor neuron and moves to the central nervous system, where TeNT again binds to gangliosides on the surface of neurons, enters the neuron by an endocytic mechanism to release the catalytic A domain into the host cell cytosol. The catalytic A domain cleaves the SNARE protein, which inhibits neurotransmitter fusion to the host cell membrane and the release of inhibitory neurotransmitter molecules. The loss of inhibitory impulses results in reflex irritability, autonomic hyperactivity, the classic large muscle spasticity and lockjaw associated with tetanus. Humans can be easily infected with tetanus if matter contaminated with C. tetani makes contact with an open wound. Once in the body, the bacterium produces TeNT, which attacks the body’s nervous system and causes spastic paralysis. This type of paralysis often causes injury to muscles, tendons, and bones as it tightens and stiffens the muscles. The development of the tetanus vaccine decreased the number of tetanus cases per year from 150,000 to 1,000. In addition, the application of TeNT in therapeutic treatments for various neurological disorders is currently being investigated. Botulism and tetanus toxins are similar, and both are organized into three domains. The N-terminal domain comprises the catalytic activity (Red, LC), the internal domain comprises the translocation domain (Green) and the C-terminal domain is the receptor binding domain (Blue, HCR). PDB: 3BTA (BoNT/A) Crystal structure of botulinum neurotoxin type A and implications for toxicity. Lacy, D.B., Tepp, W., Cohen, A.C., DasGupta, B.R., Stevens, R.C. (1998) Nat.Struct.Biol. 5: 898-902 Central Nervous System After entering an open wound on the skin, the tetanus bacterium releases TeNT, which binds to the nearest neuron in the PNS and travels inside the neuron to the CNS. The botulism bacteria behaves similarly, releasing BoNT into the PNS, yielding flaccid paralysis. The vesicles shown in blue is represent the steps for botulism. The yellow shows TeNT. The scissors are used to display the light chain of TeNT, which cleaves the SNARE protein. Peripheral Nervous System Acetycholine, a neurotransmitter, sends signals to the muscles, allowing them to contract. BoNT prevents the transmission of acetycholine. To demonstrate that TeNT needs to bind to two gangliosides to enter the neuron, an experiment was performed on human epithelial cells (HeLa), which have similar properties to neuronal cells. The detection of red light indicated successful binding of TeNT to the HeLa cell. When only one of the two gangliosides required for binding (GD3 and GM1a) was present in the cell, TeNT did not bind to the cell. But when both gangliosides were present, TeNT bound to the cell. Thus, it can be concluded that TeNT must bind to both gangliosides to bind to the cell. In the CNS, TeNT cleaves VAMP2 (SNARE protein), stopping the release of neurotransmitters. Tetanus Receptor Binding Domain This domain of TeNT allows the toxin to bind to and enter a neuron. 1fv2.pdb Tetanus and Botulism: Separated at Birth? “Opisthotonos”, in a state of a severe spasticity by Sir Charles Bell, 1809 Glycine, an inhibitory neurotransmitter, prevents the body’s muscles from having spasms. The cleavage of the SNARE protein by TeNT prevents glycine from being released. The tetanus and botulism bacteria share many characteristics in affecting the human body. Both produce neurotoxins that affect the body’s muscle function, and both bind to neurons in order to inhibit neurotransmitter activity. The only major difference between the two toxins is the part of the nervous system they affect. TeNT travels up to the central nervous system (CNS) and inhibit inhibitory neurotransmitters, causing spastic paralysis. On the other hand, the botulism neurotoxin (BoNT) remains in the peripheral nervous system (PNS) and inhibit neurotransmitters, causing flaccid (limp) paralysis. Conclusion 1.TeNT utilizes gangliosides as receptors to enter neurons. 2.TeNT causes spastic paralysis by trafficking up the nervous system from the PNS to the CNS. Once in the CNS, TeNT cleaves the SNARE protein, VAMP-2, to inhibit fusion of neurotransmitter vesicles to the plasma membrane of neurons. Outcomes of studying the structure of Tetanus Toxin 1.TeNT heavy chain receptor-binding domain (HCR) is used in tetanus vaccines to develop the body’s immunity to the toxin. 2.TeNT light chain (LC) may be useful to develop derivatives to treat neurological and non-neurological diseases. Acknowledgement: The background of the poster is adapted from wikipedia.org. A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA).