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== Structural Highlights ==
== Structural Highlights ==
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Shiga toxin (stx) has multiple classifications. The shiga toxin produced by ''Shigella dysenteriae'' is classified as stx, while shiga toxin produced by Escherichia coli can be classified as either stx1 or stx2. All three classifications very slightly in structure but have identical functions. The A sub-unit of stx1 differs from stx by only one residue: a serine in position 45 as opposed to threonine<ref name="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
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Shiga toxin (stx) has multiple classifications. The shiga toxin produced by ''Shigella dysenteriae'' is classified as stx, while shiga toxin produced by Escherichia coli is classified as either stx1 or stx2. All three classifications vary slightly in structure but have identical functions. The A sub-unit of stx1 differs from stx by one residue: a serine in position 45 as opposed to threonine<ref name="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
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Stx is a 70 kDa AB5 protein, meaning that it is composed of an A sub-unit bound to a B pentamer. A noncovalent interaction causes the association between the A and B sub-units, as the carboxy terminal tail of the A sub-unit is surrounded by the B pentamer. <scene name='68/687332/Sub-unit_a/2'>The A sub-unit</scene> is 293 amino acids long with its active site being <scene name='68/687332/Active_site_for_stx/1'>glutamic acid 167 (1R4Q)</scene>. Though the active site is a single residue, studies have shown that the first 239 residues are essential for the enzymatic activity of the A sub-unit. Residues 240-251 are essential for the A sub-unit’s translocation from the endoplasmic reticulum of an infected cell to the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>.
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Stx is a 70 kDa AB5 protein, meaning that it is composed of an A sub-unit bound to a B pentamer. A pentamer of B sub-units encircles the carboxy tail of the A sub-unit stabilized by a series of non-covalent interactions. <scene name='68/687332/Sub-unit_a/2'>The A sub-unit</scene> is 293 amino acids long with its active site being <scene name='68/687332/Active_site_for_stx/1'>glutamic acid 167 (1R4Q)</scene>. Though the active site contains a single residue, studies have shown that the first 239 residues are essential for the enzymatic activity of the A sub-unit. Residues 240-251 are essential for the A sub-unit’s translocation from the endoplasmic reticulum of an infected cell to the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>.
The A sub-unit can be broken down into A1 and A2 units. This break down occurs when a trypsin sensitive region, residues 248-251, is cleaved. This cleavage results in the only remaining link being a <scene name='68/687332/Disulfide_bridge/1'>disulfide bridge (1R4Q)</scene> between cysteine 242 and cysteine 261. If the disulfide bridge is reduced, the A1 and A2 fragments can completely separate. Interestingly, the disulfide bridge blocks the active site of the A sub-unit, so the A sub-unit is not enzymatically active unless it has been cleaved<ref name ="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
The A sub-unit can be broken down into A1 and A2 units. This break down occurs when a trypsin sensitive region, residues 248-251, is cleaved. This cleavage results in the only remaining link being a <scene name='68/687332/Disulfide_bridge/1'>disulfide bridge (1R4Q)</scene> between cysteine 242 and cysteine 261. If the disulfide bridge is reduced, the A1 and A2 fragments can completely separate. Interestingly, the disulfide bridge blocks the active site of the A sub-unit, so the A sub-unit is not enzymatically active unless it has been cleaved<ref name ="Fraser">Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200</ref>.
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<scene name='68/687332/B_sub-unit/1'>The B sub-unit (2XSC)</scene> is a symmetrical pentamer that resembles a star and can be divided into five identical monomers. Each of the monomers is 69 amino acids in length. The purpose of the B sub-unit is to bind to globotriaosylceramide (GB3) which is a glycosphingolipid that is found on the lipid rafts of endothelial cells. Each monomer of the B sub-unit has three binding sites for GB3, binding to the <scene name='68/687332/Gb3_bound_to_sub-unit_b/1'> carbohydrate portion of GB3 (1CQF)</scene>. The affinity between the B sub-unit and GB3 is incredibly low, and is actually one of the lowest recorded affinities for carbohydrate-protein interaction<ref>Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886</ref>. Some studies have shown that the level of saturation and length of the fatty acid on GB3 affects the strength of its interaction with the B sub-unit. Since the B sub-unit of stx has fifteen binding sites though, it has an incredibly high avidity<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>.
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<scene name='68/687332/B_subunit/1'>The B sub-unit (2xsc)</scene> is a symmetrical pentamer that resembles a star and can be divided into five identical monomers. Each monomer is 69 amino acids in length. The purpose of the B sub-unit is to bind to globotriaosylceramide (GB3) which is a glycosphingolipid that is found on the lipid rafts of endothelial cells. Each monomer of the B sub-unit has three binding sites for GB3, binding to the <scene name='68/687332/Gb3_bound_to_subunit_b/1'>carbohydrate portion of GB3 (1cqf)</scene>. The B sub-unit has a millimolar affinity for GB3, and is actually one of the lowest recorded affinities for carbohydrate-protein interaction<ref>Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886</ref>. Some studies have shown that the level of saturation and length of the fatty acid on GB3 affects the strength of its interaction with the B sub-unit. Since the B sub-unit of stx has fifteen binding sites though, it significantly increases the strength of the interaction.<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>.
== Function ==
== Function ==
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The pathway of stx entering a cell begins with the B sub-unit’s binding to GB3. Once this occurs, the A sub-unit disconnects from the B sub-unit and enters the cell through endocytosis. Using retrograde transport the A sub-unit passes through the Golgi apparatus and the rough endoplasmic reticulum<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref> In the rough endoplasmic reticulum, the A sub-unit is split into two parts called A1 and A2 through the cleavage of trypsin sensitive residues and the reduction of a disulfide bridge. A2 is degraded, but A1 freely enters the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>. Once in the cytosol, A1 acts as an N-glycosidase, which is an enzyme that hydrolyzes bonds that link sugars. With this enzymatic activity, A1 removes an adenine from the alpha-sarcin loop in the 28S RNA of the 60S ribosomal sub-unit<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>. The removal of the adenine prevents elongation factors from associating with the ribosomal sub-unit. Without elongation factors, the ribosome can no longer synthesize proteins, leading to cell death<ref name="Melton">Melton-Cesla, A. (2012). Shiga toxin classification structure and function. Changes, 29(2), 997–1003</ref>.
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The pathway of stx entering a cell begins with the B sub-unit’s binding to GB3. Once this occurs, the A sub-unit disconnects from the B sub-unit and enters the cell through endocytosis. Using retrograde transport the A sub-unit passes through the Golgi apparatus and the rough endoplasmic reticulum<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref> In the rough endoplasmic reticulum, the A sub-unit is cleaved into two parts, A1 and A2, through the cleavage of trypsin sensitive residues and the reduction of a disulfide bridge. A2 is degraded, but A1 freely enters the cytosol<ref name="Sandvig">Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943</ref>. Once in the cytosol, A1 acts as an N-glycosidase, hydrolyzing bonds that link sugars. With this enzymatic activity, A1 removes an adenine from the alpha-sarcin loop in the 28S RNA of the 60S ribosomal sub-unit<ref name="Melton">Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web</ref>. The removal of the adenine prevents elongation factors from associating with the ribosomal sub-unit. Without elongation factors, the ribosome can no longer synthesize proteins, leading to cell death<ref name="Melton">Melton-Cesla, A. (2012). Shiga toxin classification structure and function. Changes, 29(2), 997–1003</ref>.
== Disease and Pathogenesis ==
== Disease and Pathogenesis ==
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The shiga toxin is produced by ''Shigella dysenteriae'' and certain strains of ''Escherichia coli''. ''S. dysenteriae'' is a gram negative bacterium that causes disease in its host by entering into intestinal epithelial cells. It has a very low infectious dose of only ten to one hundred cells, and is contracted through the consumption of contaminated water or food. One it begins replicating in the intestines, its proliferation and secretion of stx erodes the intestinal lining and leads to dysentery<ref>Zaidi, Mussaret Bano, and Teresa Estrada-García. "Shigella: A Highly Virulent and Elusive pathogen." Current Tropical Medicine Reports 1.2 (2014): 81-87</ref>. Those with dysentery can simply have diarrhea as a symptom, but if the infection progresses, it can lead to hemorrhagic colitis (bloody stool) or hemolytic uremic syndrome (HUS). HUS is characterized as acute renal failure, thrombocytopenia, and microangiopathic anemia<ref>Kaper, James B. and Alison D. O’Brien. “Overview and Historical Perspectives.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0028–2014. PMC. Web</ref>.
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The shiga toxin is produced by ''Shigella dysenteriae'' and certain strains of ''Escherichia coli''. ''S. dysenteriae'' is a gram negative bacterium that causes disease in its host by entering into intestinal epithelial cells. It has a very low infectious dose of only ten to one hundred cells, and is contracted through the consumption of contaminated water or food. Once in intestines, the bacteria's proliferation and secretion of stx erodes the intestinal lining and leads to dysentery<ref>Zaidi, Mussaret Bano, and Teresa Estrada-García. "Shigella: A Highly Virulent and Elusive pathogen." Current Tropical Medicine Reports 1.2 (2014): 81-87</ref>. Those with dysentery can simply have diarrhea as a symptom, but if the infection progresses, it can lead to hemorrhagic colitis (bloody stool) or hemolytic uremic syndrome (HUS). HUS is characterized as acute renal failure, thrombocytopenia, and microangiopathic anemia<ref>Kaper, James B. and Alison D. O’Brien. “Overview and Historical Perspectives.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0028–2014. PMC. Web</ref>.
As stated previously, stx enters cells through its binding to GB3, but curiously many intestinal cells do not express GB3. Some researchers hypothesize it enters the cells through macropinocyosis, but when macropinocytotic function is removed from cells, stx still manages to enter through an as yet undefined mechanism.
As stated previously, stx enters cells through its binding to GB3, but curiously many intestinal cells do not express GB3. Some researchers hypothesize it enters the cells through macropinocyosis, but when macropinocytotic function is removed from cells, stx still manages to enter through an as yet undefined mechanism.
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The genes necessary to produce the toxin are interestingly not native to ''S. dysenteriae''. The genes for stx are carried by prophages: bacteria that have had viral DNA inserted into their genome. During certain conditions, the phage genes are expressed leading to the production of more bacteriophages as well as the shiga toxin. When the bacteria undergo lysis, the bacteriophages and stx are released. It may seem as if there is no benefit for ''S. dysenteriae'' to carry the viral DNA, but studies have shown that while it is in the lysogenic cycle S. dysenteriae has increased acid tolerance and motility<ref>Krüger, Alejandra, and Paula María Alejandra Lucchesi. "Shiga toxins and stx-phages: highly diverse entities." Microbiology (2014): mic-0</ref>.
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The genes necessary to produce the toxin are interestingly not native to ''S. dysenteriae''. The genes for stx are carried by prophages: bacteria that have had viral DNA inserted into their genome. During certain conditions, the phage genes are expressed leading to the production of more bacteriophages as well as the shiga toxin. When the bacteria undergo lysis, bacteriophage and stx are released. It may seem as if there is no benefit for ''S. dysenteriae'' to carry the viral DNA, but studies have shown that while it is in the lysogenic cycle S. dysenteriae has increased acid tolerance and motility<ref>Krüger, Alejandra, and Paula María Alejandra Lucchesi. "Shiga toxins and stx-phages: highly diverse entities." Microbiology (2014): mic-0</ref>.
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This Sandbox is Reserved from 15-Jan-2015, through 30-May-2015 for use in the course "Biochemistry" taught by Jason Telford at the Maryville University. This reservation includes Sandbox Reserved 977 through Sandbox Reserved 986.
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Shiga Toxin

Crystal Structure for Shiga Toxin (1R4Q)

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References

  1. ↑ 1.0 1.1 Fraser, M. E., Fujinaga, M., Cherney, M. M., Melton-Celsa, A. R., Twiddy, E. M., O’Brien, A. D., & James, M. N. G. (2004). Structure of shiga toxin type 2 (Stx2) from Escherichia coli O157:H7. The Journal of Biological Chemistry, 279(26), 27511–27517. doi:10.1074/jbc.M401939200
  2. ↑ 2.0 2.1 Sandvig, K., & van Deurs, B. (2000). Entry of ricin and Shiga toxin into cells: molecular mechanisms and medical perspectives. The EMBO Journal, 19(22), 5943–5950. doi:10.1093/emboj/19.22.5943
  3. ↑ Jacobson, J. M., Yin, J., Kitov, P. I., Mulvey, G., Griener, T. P., James, M. N. G., … Bundle, D. R. (2014). The crystal structure of Shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor. Journal of Biological Chemistry, 289(2), 885–894. doi:10.1074/jbc.M113.518886
  4. ↑ 4.0 4.1 4.2 4.3 Melton-Celsa, Angela R. “Shiga Toxin (Stx) Classification, Structure, and Function.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0024–2013. PMC. Web
  5. ↑ Zaidi, Mussaret Bano, and Teresa Estrada-García. "Shigella: A Highly Virulent and Elusive pathogen." Current Tropical Medicine Reports 1.2 (2014): 81-87
  6. ↑ Kaper, James B. and Alison D. O’Brien. “Overview and Historical Perspectives.” Microbiology spectrum 2.2 (2014): 10.1128/microbiolspec.EHEC–0028–2014. PMC. Web
  7. ↑ Krüger, Alejandra, and Paula María Alejandra Lucchesi. "Shiga toxins and stx-phages: highly diverse entities." Microbiology (2014): mic-0
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