Tetanus is a severe disease caused by the bacterium Clostridium tetani, characterized by muscle spasms, notably starting from the jaw. The neurotoxin released affects the central nervous system, leading to persistent motor neuron signaling that causes muscle contractions. Clostridium tetani is highly lethal, with significant mortality rates, yet reliable diagnostic tests are lacking, complicating treatment.

1. Tetanus Toxin Mechanism
Dariyus Z Kabraji
MSc: 1 Roll No: 18
Department of Biotechnology
Bacteriology Assignment

2. Tetanus
• Tetanus is one of the most basic fatal diseases in existence.
• It is caused by infection of the victim with Clostridium tetani.
• Also known as lockjaw, it is characterised by muscular spasms.
• In the most common type the spasms begin in the jaw and
then progresses to the rest of the body. These spasms usually
last a few minutes each time and occur frequently for three to
four weeks.
• Symptoms include bone fractures, fever, headache, trouble
swallowing, high blood pressure, and a fast heart rate.
• The incubation period is approximately eight days. In theory,
the farther the injury site is from the central nervous system,
the longer the incubation period, and the less severe are the
symptoms experienced.

3. Statistics
• Clostridium tetani is considered to be the deadliest
bacteriological pathogen in existence, second only to
Clostridium botulinum.
• The more frequent cases like neonatal tetanus resulted
in over 59000 infant mortalities in 2008 alone.
• The United States alone reports over 30 cases of
infection each year.
• Lack of a proper diagnosis test makes Tetanus all the
more potent a killer.
• Alongside the neurotoxin, C. tetani also produces the
exotoxin tetanolysin, a hemolysin, that causes
destruction of tissues.

4. Mechanism of Infection
• C. tetani usually enter the body through an open
wound, leading to spore germination under
anaerobic conditions.
• Once spore germination has occurred, toxins are
released into the bloodstream and lymphatic
system. These toxins act at several locations
within the central nervous system, interfering
with neurotransmitter release and blocking
inhibitor impulses. Such disruptions lead to
uncontrollable muscle contractions.

6. The Neurotoxin
• TeNT is the toxin complex affecting the nervous system of the victim.
• It is also known as Tetanospasmin or as the Spasmogenic toxin.
• It’s course of action involves:
1. Specific binding in the periphery neurons
2. Retrograde axonal transport to the central nervous system (CNS) inhibitory
interneurons (Movement toward the cell body is called retrograde transport
and movement toward the synapse is called anterograde transport)
3. Transcytosis from the axon into the inhibitory interneurons
4. Temperature and pH mediated translocation of the light chain into the cytosol
5. Reduction of the disulphide bond between the light and heavy chain
6. Cleavage of synaptobrevin / VAMP (Vesicle associated membrane proteins
(VAMP) are a family of SNARE proteins with similar structure, and are mostly
involved in vesicle fusion.
7. The best studied SNAREs are those that mediate docking of synaptic vesicles
with the presynaptic membrane in neurons. These SNAREs are the targets of the
bacterial neurotoxins responsible for botulism and tetanus.)

7. Clathrin Dependant Mechanism
• Neurons have adapted their endocytic pathways to
better adjust to their specific requirements. Thus,
synaptic vesicle (SV) recycling is the predominant form
of neuronal endocytosis at the presynaptic terminal,
whereby the fast fusion of neurotransmitter-containing
vesicles is coordinated with an efficient mechanism of
membrane recovery, which involves clathrin.
• In neurons, clathrin-independent routes have also been
documented, although the physiological relevance of
endocytosis via caveolae has been questioned in these
cells because several of the caveolin isoforms found in
other tissues are not detectable.

8. Course of Action
• Tetanus toxin is composed of a heavy chain and light chain, which
are attached by a disulphide bond.
• Tetanus toxin fragment C (TeNT-FC) is a 47-kDa fragment on the
heavy chain molecule that contains the ganglioside-binding domain.
• TTFC attaches to gangliosides on the peripheral nerves, and as a
result, the toxin is internalized. Through trans-synaptic spread, the
toxin can spread to the central nervous system.
• The light chain contains a zinc metalloprotease domain which can
cleave proteins that facilitate synaptic vessel fusion with the plasma
membrane of the neuron – namely, the integral protein
synaptobrevin. As a result, the neurotransmitter g-aminobutyric
acid (GABA) is blocked from reaching the synaptic cleft, and the
excitation of motor neurons persists. Persistent neuron signalling
leads to the motor spasms seen in a typical tetanus patient

9. The Neurotoxin’s Structure

10. Abstract
• Ligand–receptor complexes are internalized by a variety of endocytic
mechanisms. Some are initiated within clathrin-coated membranes, whereas
others involve lipid microdomains of the plasma membrane. In neurons, where
alternative targeting to short- or long-range trafficking routes underpins the
differential processing of synaptic vesicle components and neurotrophin
receptors, the mechanism giving access to the axonal retrograde pathway
remains unknown. To investigate this sorting process, we examined the
internalization of a tetanus neurotoxin fragment (TeNT HC), which shares axonal
carriers with neurotrophins and their receptors.
• Previous studies have shown that the TeNT HC receptor, which comprises
polysialo-gangliosides, resides in lipid microdomains. We demonstrate that TeNT
HC internalization also relies on a specialized clathrin-mediated pathway, which
is independent of synaptic vesicle recycling. Moreover, unlike transferrin uptake,
this AP-2–dependent process is independent of epsin1. These findings identify a
pathway for TeNT, beginning with the binding to a lipid raft component (GD1b)
and followed by dissociation from GD1b as the toxin internalizes via a clathrin-
mediated mechanism using a specific subset of adaptor proteins.

11. Introduction
• Endocytosis is essential for a variety of cellular functions, including the internalization of
nutrients and communication among cells, or between cells and their environment.
• Internalized molecules must be precisely sorted to their final cellular destinations to fulfil
their specific function.
• Distinct endocytic pathways have been described to date, including clathrin- dependent
endocytosis and caveolae-mediated uptake, which remain the two best-characterized
mechanisms of internalization.
• Transport vesicles bud off as coated vesicles, which have a distinctive cage of proteins
covering their cytosolic surface. Before the vesicles fuse with a target membrane, they
discard their coat, as is required for the two cytosolic membrane surfaces to interact directly
and fuse. The coat performs two main functions. First, it concentrates specific membrane
proteins in a specialized patch, which then gives rise to the vesicle membrane. In this way, it
selects the appropriate molecules for transport. Second, the coat moulds the forming vesicle.
Coat proteins assemble into a curved, basketlike lattice that deforms the membrane patch
and thereby shapes the vesicle. This may explain why vesicles with the same type of coat
often have a relatively uniform size and shape.
• Clathrin-coated vesicles, for example, mediate transport from the Golgi apparatus and from
the plasma membrane, whereas COPI- and COPII-coated vesicles most commonly mediate
transport from the ER and from the Golgi cisternae

12. Materials
• Reagents: Sulfo-NHS-SS-biotin, EZ-link–activated
maleimide-HRP (HRP is horseradish peroxidase, an
amplifier that’s used by coupling it with TeNT)
• Plasmids: encoding dynamin k44A, epsin 1R63L H73L,
and AP180 C-terminal mutants
• Antibodies: 9E10, X22, 12CA5, 69.1, epsin1, IgG3
mouse monoclonal antibody MOG1
• Neurotoxin: TeNT HC
• Miscellaneous: Edetic Acid (EDTA), PBS, ConA–
Sepharose, α-methylmannoside, NiNTA-agarose, 20
mM Hepes-NaOH, 150 mM NaCl, and 500 mM
imidazole.

13. Methods
• Protein Labelling
• Microinjection and Internalization assay
• Immunofluorescence
• Confocal microscopy

14. Observation
• TeNT HC enters clathrin-coated structures in
MNs
• MNs were incubated with HRP–TeNT HC for
45 min on ice, and then chased for 45 min at
12 or 18°C

15. Observation
• A&b =12 degrees
• C= 18 degrees
• TeNT HC internalization in MNs is independent
of presynaptic activity
• TeNT HC uptake is dynamin-dependent

17. • Transferrin uptake is mediated by a classical clathrin-dependent
internalization route occurring in soma and dendrites.
• TeNT HC exploits a pathway requiring lipid rafts and the clathrin
machinery, which is distinct from aforementioned routes of
internalization.
• At the NMJ, TeNT HC binds to a lipid–protein receptor complex
containing the ganglioside GD1b. TeNT HC is then laterally sorted
into CCPs and, during this sorting event, GD1b is excluded from
the toxin receptor c omplex.
• Internalization of TeNT HC is dependent on dynamin, AP-2, and
AP180, but does not require epsin1. Once internalized, TeNT HC is
targeted to a stationary early sorting compartment (L akadamyali
et al., 2006), to which other endocytic routes may converge. This
early sorting compartment is functionally coupled to the axonal
retrograde transport pathway.

18. References
• Katrin Deinhardt, Otto Berninghausen, Hugh J. Willison,
Colin R. Hopkins, and Giampietro Schiavo; Tetanus
toxin is internalized by a sequential clathrin-dependent
mechanism initiated within lipid microdomains and
independent of epsin1; The Journal of Cell Biology, Vol.
174, No. 3, July 31, 2006 459–471
• Farrar JJ, LM Yen, T Cook, N Fairweather, N Binh, J
Parry, CM Parry. 2000; Neurological Aspects of Tropical
Disease: Tetanus. Journal of Neurology, Neurosurgery,
and Psychiatry; 69: 292-301.
• Albert’s Molecular Biology of the Cell, 5th edition,
Chapter 13: Intracellular vesicular traffic; 749-812