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Diphtheria toxin

Diphtheria Toxin is an exotoxin produced by the organism Corynebcterium diphtheria which has been infected by a bacteriophage that contains the Diphtheria toxin gene. The toxin is the causative agent of diphtheria. Symptoms for Diphtheria can range from a sore throat with low-grade fever and an adherent pseudomembrane of the tonsils, pharynx, or nose to infected skin lesions which lack a characteristic appearance.^[1] The toxin is distributed to distant organs by the circulatory system and may cause paralysis and congestive heart failure.^[2] The toxin attacks and kills eukaryotic cells by inactivating the Elongation factor (EF-2) in translation. EF-2 allows for translocation of the peptidyl-tRNA from the A-site to the P-site, which in turn frees the A site for another aminoacyl-tRNA to bind. By inactivating the elongation factor 2 during translation the protein being made cannot be completed and therefore becomes nonfunctional. The toxin is in a class of A-B which includes cholera toxin, Escherichia coli heat labile enterotoxin, Pseudomonas aeruginosa exotoxin A, tetanus, botulinum neurotoxins, and Shiga toxin. A-B class is characterized by two functionally distinct components such as component A is the catalytic components while component B does the receptor binding function.

History

Scientist have made efforts since at least the 1740's to find the caustion and cure for diphtheria toxin. It wasn't until Koch's devolpment of medical microbiology and Koch's postulates that it was even possible for progression towards understanding the molecular causes. It was later in Koch's laboratory that Friedrich Loeffler isolated a bacteria from a patient that died from Diphtheria toxin. Koch and Friedrich later inoculated 23 guinea pigs with the isolated bacteria and they all died in two to five days. Loeffler had another observation that only the isolated bacteria from the pigs were growing at the site of inoculation. Therefore, he came to the conclusion that the bacteria isolated was the causation of the Diphtheria toxin. In Pasture institute in Paris, both Emile Roux and Alexandre Yersin were able to isolate the toxin by growing a pure culture of Diphtheria bacilli and forcing them through a porcelain filter. This allowed to obtain no bacteria and only the toxin. The toxin was injected into laboratory animals and caused the same symptoms of Diphtheria bacilli. It wasn't until sixty-five years later that protein crystals were produced and the structure obtained. Emil von Behring published a paper with the discovery of diphtheria antitoxin one week after Koch found out the resistance gained by vaccination from Diphtheria bacilli. This lead to elimination of the disease diphtheria in developing countries.

Von Behring received a Nobel Prize for the antitoxin which later would be known as antibodies against Diphtheria toxin. The next vaccine made was one mixed of toxin and antitoxin. The antitoxin relieved the harmful effects of the toxin which was the real vaccine. This was later replaced in 1923 with a vaccine of a formaldehyde-treated Diphtheria toxin. Today in developing countries people get routine vaccinations so that diphtheria is unknown.

Structure

Diphtheria toxin is a protein made of 535 amino acids and is 58kDa in weight. The proenzyme(zymogen) must be cleaved into fragments A & B and reduced in order for the toxicity gene to be turn on. In each fragment there are two . There are also three domains C,T, and R that have different functions. The first crystal structure of the toxin was obtained in 1992 by x-ray crystallography. However, the structure was a dimer and generated by freezing the protein. The dimer is non-toxic but becomes a toxic monomer at neutral pH by dissociation. The of the molecule is made up of 24 helices, 31 beta sheets, and turns between each of them which adds up to be 535 residue protein. This makes for a mixture of that allow for the seondary structure. The purple portion of the molecule is the polar ares while the gray was the hydrophobic areas.

• is about molecular mass of 21kDa and is the catalytically active portion of the protein.
• is about 37kDa and contains the receptor binding & translocation portions of the protein.

The two fragments are then separated into three domains C, T, and R which correspond to three major functions of the toxin.

• In fragment A contains domain C which contains the and does the catalysis for the protein. It contains eight β strands and seven α helices.

• The T domain is found in fragment B and is the translocation portion of the protein. The structure is made up of three layers of α helices. The outer layer contains 3 helices that are rich in polar residues that allow for the molecule to maintain the T domain as well as keeping the toxin soluble in neutral pH.

• Another domain in fragment B is domain R which has the for the protein. The domain consist mostly of β sheets and changes conformation when interacting with cell membranes. In the R domain both Lsy516 and Phe530 play a role in recognizing receptors on cell surfaces.

In the mechanism for Diphtheria toxin the R Domain binds to which are NAD⁺ which can sometimes be switched with ApUp. The molecule ApUp is bound to a cleft on the front side of the C domain.

^[3]

Mechanism

The mechanism begins by the R domain recognizing the target cells by the molecules on the cells surface. A prominent β-hairpin loop of the R domain binds to the surface and allows for a docking station. The ligand that is typically bound on the cell surface is pro-HB-EFG. Binding leads to receptor mediated endocytosis. The T domain translocates the C domain into the cytosol of the cell. The organism Corynebacterium diphtheriae first secretes the toxin the loop between the C and T domain has to be nicked in order for the toxicity to be turned on. There is one surface protease named furin that is known to nick the area but it is unclear if any other protein helps the process. This process allows for the C domain to dissociate from the T domain after transported(translocated) into the cytosol. This is where the disulfide bonds become reduced. After this happens a drop in pH leads to the formation of the dimer to become an open monomer form. The reduction of the disulfide bonds is the rate determining step of the toxin entrying the cell.

Once entried into the cytosol, the C domain does ADP-ribosylation of EF-2 at diphthamide, a posttranslationally modified histidine residue. This shuts down all protein synthesis and kills the cell. The K_cat/K_M of the reaction is about 10⁸ min^-1 M^-1. The figure below shows the reaction happening at the C domain. The C domain binds to the ligand NAD⁺ to begin the reaction. The C domian does have an inhibitor and that is ApUp which is near in the C domain.

figure 3: Mechanism of inactivation of elongation factor 2 (EF2) by diphtheria toxin ^[4]

Medical Implications & Possible Application

Diphtheria toxin is the causative agent of the disease Diphtheria. The lethal dose for humans and other susceptible eukarya is .1μg of toxin per kg of body weight^[5] A vaccine has been made and is routinely used in first world countries as a basic immunization. The vaccine is made up of formaldehyde-treated Diphtheria toxin. However, Diphtheria toxin has been used is many other medical applications such as the drug Denileukin diftitox and cancer suppressors. Denileukin diftitox is a protein that combines Diphtheria toxin and Interleukin 2. In some Leukemias and Lymphomas cells express Interleukin receptors in which the drug can bind to and release the toxin within the cells.

Another application that Diphtheria toxin can be used for is a cancer suppressor. In terms of the R domain of the protein which as we know binds to pro-HB-EFG receptors of the surface of cells and is translocated into the cell by endocytosis. Some scientist have made a non-toxic mutant of diphtheria toxin CRM197 which is being used to block the signaling activity of the receptor. In some cancers such as ovarian cancer, pro-HB-EFG is overexpressed and blocking the signal has shown promise in suppressing the effects of pro-HB-EFG on cancer development.^[6] Another example, is called immunotoxins which change the R domain to recognize a specific cell surface and kill that cell. This is being tested against different cancer cell types and has been successful.

References

• Parker, M. (1996). Protein toxin structure. (1 ed., pp. 25-43). Georgetown: R.G. Landes Company.

• Bennett, M., & Eisenberg, D. (2009, febuary 24). The refined structure of monomeric diphtheria toxin at 2.3 angstroms resolution. Retrieved from http://www.pdb.org/pdb/explore/explore.do?

• Stephen, J., & Pietrowski, R. (1981). Bacterial toxins. (1 ed., p. 11). Washington: Van Nostrand Reinhold Co. Ltd.

• Oram , D., & Holmes, R. (2006). Diphtheria toxin. (3 ed., pp. 245-252). Burlington, San Diego, London: Academic Press publications.

• Diphtheria toxin. (2011, November 25). Retrieved from <http://en.wikipedia.org/wiki/Diphtheria_toxin>

• Murphy JR (1996). "Corynebacterium Diphtheriae: Diphtheria Toxin Production". In Baron S et al.. Medical microbiology (4 ed.). Galveston, Texas: Univ. of Texas Medical Branch. ISBN 0-9631172-1-1.

Footnotes