Clostridium tetani and Tetanus: Difference between revisions
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===Cell Entry=== | ===Cell Entry=== | ||
Tetanospasmin must enter nerve cells in order to reach its primary site of action and produce clinical tetanus disease. The toxin can have some effect on motor neurons near the site of infection, but the main syndrome is produced after TeTx reaches the spinal cord. Once the toxin has entered neurons near the site of infection or elsewhere in the peripheral nervous system, it can move retrogradely within axons, to the cell body of the neuron, then subsequently to other neurons, and thus reach the spinal cord via intra-axonal transport (Erdmann 1975). | |||
The TeTx protein is comprised of two components, both of which are necessary for intoxication but have different functions in pathogenesis. TeTx is initially synthesized as a ~150-kDa chain, but is subsequently cleaved into a two-part chain linked by a single disulfide bond (Chen 2009). The final two components are the light chain (LC; ~50 kDa) and the heavy chain (HC; ~100 kDa). The LC is the active constituent of TeTx, but the HC is required for entry into the cell, where LCs function to cause disease (Binz 2009). | |||
The HCs of TeTx interact with gangliosides on the surface of neuronal membranes. Gangliosides are sialic acid-containing glycosphingolipids that may be involved in signal transduction and are found in the greatest concentrations on the outer layers of membranes in the nervous system (Yu 2008). In preparations of mouse neurons where production of gangliosides has been inhibited there is no sign of TeTx binding to cells and the toxin does not prevent the release of neurotransmitter from those cells (Williamson 1999). Just as TeTx is divided into two domains, HC can also be divided into two functionally distinct domains. The amino-terminal domain HN is responsible for translocating the LC across the plasma membrane, whereas the carboxyl-terminal domain HC is responsible for the binding of TeTx to gangliosides on neurons (Rummel 2003). | |||
The HC domain can be broken down even further into the HCN and HCC subunits. The HCC fragment has been shown to bind the gangliosides and helps in retrograde transport along the axon so that TeTx can reach the spinal cord (Rummel 2003). The HCN domain does not bind gangliosides (Figueiredo 1995) and its ultimate role remains unclear. The HCC domain has two important binding sites associated with it. These pockets are termed the lactose binding site and the sialic acid binding site (Binz 2009). In one set of experiments, researchers mutated these binding pockets by changing certain amino acids. When only the sialic acid binding site was mutated, toxicity was reduced by almost 99%; toxicity levels were almost undetectable after mutation of the lactose site (Rummel 2003). Furthermore, the experiments were performed on GT1b-type gangliosides, which have the highest affinity for TeTx. Another study used gangliosides of the type GM1a, which binds only the lactose pocket, and GD3, which binds only the sialic acid pocket, in order to explicate the role of these pockets in TeTx binding. They confirmed again that a mutated HCC fragment lack one pocket or the other did not bind TeTx. In the presence of only one of the gangliosides—either GM1a or GD3—toxin was unable to enter the cell (Chen 2009). The results from both investigations show that both binding sites within the HCC domain are necessary for TeTx to enter the cell. In addition, it appears as though two separate gangliosides are needed to bind HCC. | |||
Once the HC domain has bound to the gangliosides, the HN domain then allows the LC to cross into the cytoplasm of the cell, where it can act on neurotransmitter vesicles. It is currently unknown how exactly HN helps the LC gain access to the cytoplasm. Models have been proposed for BoNT, which is structurally similar to TeTx. In these models, the low pH of the endosome may cause a structural change in the HC causing the HN domain to insert into the endosome membrane and form a channel (Brunger 2007). The HN channel may also act as a chaperone, unfolding the LC, pulling it across the endosomal transmembrane channel, and then refolding it in the cytoplasm of the neuron (Binz 2009). | |||
===Mechanism of Action within the Cell=== | ===Mechanism of Action within the Cell=== | ||
Revision as of 02:03, 25 April 2011
Introduction
By Tyler Stearns
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The Tetanus Toxin: Genetics and Mechanism of Action
Genetics
The genome of C. tetani consists of a single 2,799,250-bp chromosome with 2,372 open reading frames (ORFs); the tetanus toxin (TeTx) is encoded on a 74,082-bp plasmid containing 61 ORFs (Brüggemann 2003). The genus Clostridium is a member of the Firmicutes phylum, which are known for a low G-C content (S & F 2011). The C. tetani chromosome has a G-C content of 28.6%, while the TeTx-encoding plasmid pE88 only has a G-C content of 24.5%. The G-C ratio is relatively stable in the main chromosome, indicating a lack of recent horizontal gene transfer (Brüggemann 2003).
The plasmid pE88 holds the genes for TeTx (tetX) and its direct transcriptional regulator TetR. The tetR gene is located just upstream from tetX (Brüggemann 2005). TetR was thought to possibly be a positive regulatory of toxin expression, but current research has shown that TetR is in fact a sigma factor of a subgroup unique to clostridial species. This research found that TetR only associated with target DNA in the presence of the RNA polymerase core enzyme, bound to the core enzyme, and triggered transcription of the target DNA at the promoter in vitro (Raffestin 2005). Other regulatory genes are present on the plasmid. CTP05, CTP10, and CTP11 are sigma factor-like proteins, and CTP21 and CTP22 form a two-component system of unknown regulatory function. The three sigma-like proteins do not appear to be involved in the regulation of toxin production like TetR (Brüggemann 2005; Raffestin 2005).
Other virulence factors may be present on pE88, in addition to the primary tetanus toxin. The 114-kDa collagenase ColT is also encoded on the plasmid (Brüggemann 2003). Collagenase is an enzyme that degrades collagen, which makes up almost 25 to 33% of the total protein in mammalian organisms (Harrington 1996). Thus, ColT may help destroy tissue in an infected host.
The main chromosome of C. tetani also possesses potential virulence factors. ORFs for tetanolysin O, hemolysin, and fibronectin-binding proteins, as well as genes for surface-layer (S-layer) proteins have been identified (Brüggemann 2003). Tetanolysin, discussed below, is one of the two exotoxins produced by C. tetani, in addition to TeTx. The S-layer has been characterized as a molecular sieve or as a possible defense against parasites. However, it also has been implicated in host cell adhesion or as a possible mechanism of evading host immune systems in another pathogenic clostridial species, C. difficile (Spigaglia 2011).
The origin of the plasmid pE88 is still unclear. Over 50% of the ORFs on the plasmid are unique to C. tetani (Brüggemann 2003). Toxin genes are currently—or were in their evolutionary history—part of a flexible clostridial gene pool. Many of the toxin genes in pathogenic Clostridium species are on plasmids or capable of transduction by phages (Brüggemann 2005). C. botulinum, which produces various forms of the botulinum neurotoxin (BoNT), has been shown to transfer its toxin-encoding plasmid by conjugation. In one study, a tagged BoNT-encoding plasmid was transferred from one strain of C. botulinum to another. Bacteriophages for transduction were not observed, and gene transfer was not inhibited by DNase, ruling out transformation of free-floating DNA (Marshall 2010). The epsilon-toxin plasmids in C. perfringens Type D have also been shown to be transferable to other cells by conjugation (Hughes 2007). Given this evidence, and the fact TeTx is quite similar to BoNT, it seems highly probably that the TeTx plasmid was acquired via some form of horizontal gene transfer, though it has clearly undergone its own divergent evolution since that point.
Infection
Tetanus infections most commonly occur after deep-tissue puncture wounds that are exposed to C. tetani. Contamination of the wound often involves contact with soil, fecal matter, or rusty metal that contains C. tetani (Cook 2001; Campbell 2009). C. tetani is an obligate anaerobe. The conditions in a wound are particularly well suited to the bacteria's anaerobic needs (Campbell 2009). Tetanolysin is one of the two exotoxins excreted by C. tetani. Tetanolysin damages tissue surrounding an infection, optimizing conditions within the wound (Cook 2001; Campbell 2009). Bacteria grow and ferment in the wound, releasing in small quantities the actual causative agent of tetanus disease, the tetanospasmin protein (also referred to as tetanus toxin, TeTx, or TeNT). The toxin is mainly released during the stationary phase of growth and a significant majority of the toxin is not freed until a cell lyses, releasing its contents into the body of the host (Mellanby 1981). TeTx is distributed to motor neurons and in the bloodstream to other pre-synaptic nerve terminals in the peripheral nervous system (Kerr 1979).
Cell Entry
Tetanospasmin must enter nerve cells in order to reach its primary site of action and produce clinical tetanus disease. The toxin can have some effect on motor neurons near the site of infection, but the main syndrome is produced after TeTx reaches the spinal cord. Once the toxin has entered neurons near the site of infection or elsewhere in the peripheral nervous system, it can move retrogradely within axons, to the cell body of the neuron, then subsequently to other neurons, and thus reach the spinal cord via intra-axonal transport (Erdmann 1975).
The TeTx protein is comprised of two components, both of which are necessary for intoxication but have different functions in pathogenesis. TeTx is initially synthesized as a ~150-kDa chain, but is subsequently cleaved into a two-part chain linked by a single disulfide bond (Chen 2009). The final two components are the light chain (LC; ~50 kDa) and the heavy chain (HC; ~100 kDa). The LC is the active constituent of TeTx, but the HC is required for entry into the cell, where LCs function to cause disease (Binz 2009).
The HCs of TeTx interact with gangliosides on the surface of neuronal membranes. Gangliosides are sialic acid-containing glycosphingolipids that may be involved in signal transduction and are found in the greatest concentrations on the outer layers of membranes in the nervous system (Yu 2008). In preparations of mouse neurons where production of gangliosides has been inhibited there is no sign of TeTx binding to cells and the toxin does not prevent the release of neurotransmitter from those cells (Williamson 1999). Just as TeTx is divided into two domains, HC can also be divided into two functionally distinct domains. The amino-terminal domain HN is responsible for translocating the LC across the plasma membrane, whereas the carboxyl-terminal domain HC is responsible for the binding of TeTx to gangliosides on neurons (Rummel 2003).
The HC domain can be broken down even further into the HCN and HCC subunits. The HCC fragment has been shown to bind the gangliosides and helps in retrograde transport along the axon so that TeTx can reach the spinal cord (Rummel 2003). The HCN domain does not bind gangliosides (Figueiredo 1995) and its ultimate role remains unclear. The HCC domain has two important binding sites associated with it. These pockets are termed the lactose binding site and the sialic acid binding site (Binz 2009). In one set of experiments, researchers mutated these binding pockets by changing certain amino acids. When only the sialic acid binding site was mutated, toxicity was reduced by almost 99%; toxicity levels were almost undetectable after mutation of the lactose site (Rummel 2003). Furthermore, the experiments were performed on GT1b-type gangliosides, which have the highest affinity for TeTx. Another study used gangliosides of the type GM1a, which binds only the lactose pocket, and GD3, which binds only the sialic acid pocket, in order to explicate the role of these pockets in TeTx binding. They confirmed again that a mutated HCC fragment lack one pocket or the other did not bind TeTx. In the presence of only one of the gangliosides—either GM1a or GD3—toxin was unable to enter the cell (Chen 2009). The results from both investigations show that both binding sites within the HCC domain are necessary for TeTx to enter the cell. In addition, it appears as though two separate gangliosides are needed to bind HCC.
Once the HC domain has bound to the gangliosides, the HN domain then allows the LC to cross into the cytoplasm of the cell, where it can act on neurotransmitter vesicles. It is currently unknown how exactly HN helps the LC gain access to the cytoplasm. Models have been proposed for BoNT, which is structurally similar to TeTx. In these models, the low pH of the endosome may cause a structural change in the HC causing the HN domain to insert into the endosome membrane and form a channel (Brunger 2007). The HN channel may also act as a chaperone, unfolding the LC, pulling it across the endosomal transmembrane channel, and then refolding it in the cytoplasm of the neuron (Binz 2009).
Mechanism of Action within the Cell
Clinical Tetanus
Symptoms
Prevention and Treatment
Vaccination
Treating the Infection
Treating the Symptoms
Conclusion
Include some current research, with at least one figure showing data.
References
Edited by student of Joan Slonczewski for BIOL 238 Microbiology, 2011, Kenyon College.
