Diphtheria Toxin: A Model for Translocation

By SOVANN UCH

In living cells the plasma membrane serves many functions including protecting the cell, synthesizing ATP, providing receptor sites, cell recognition, and translocation (Mekada, 1979). Inside of the cell there are endosomal vesicles that facilitate the translocation of macromolecules from the outside of the cell to the inside of the cell. Cells have receptors for the uptake in these external macromolecules by endocytosis. Once a protein has translocated through the membrane and finds its target receptor, it may then cause its effect inside the cell (Karp, 331).

Corynebacterium diphtheriae is a gram-positive bacillus with an irregular shape. Bacteria that stain gram-positive have a thicker cell wall composed of peptidoglycan. It is this thicker cell wall on the microbe that causes it to appear purple under the microscope. Most bacilli are spheroid in shape, but this microbe’s shape resembles the alphabet letters V or Y. It is ubiquitous in plants and animals. It will normally colonize the skin, upper respiratory tract, the gastrointestinal tract and the urogenital tract (Murray, 1998). These microbes are extremely opportunistic pathogens.

The modes of transmission for these bacteria are from person to person by respiratory droplets or skin contact. C. diphtheriae does not enter the blood stream directly to cause its disease. Humans are the only known carriers of the disease. In 1980, in the United States there were less than 5 reported cases of this disease. Diphtheria targets worldwide populations were vaccination programs are not administered. People who are unvaccinated or immunocompromised will most likely be at risk.

The symptoms of this disease include being asymptomatic, having mild respiratory symptoms and even fatality in non-immunized people. The organism can multiply rapidly on the respiratory tract; after exposure, 2 to 6 days later there is localized death to the epithelial cells in the pharynx and adjacent cells.

Administration of diphtheria antitoxin is necessary to neutralize the exotoxin by preventing the exotoxin from binding with host cells. Penicillin or erythromycin is used to eliminate the C. diphtheriae, which eliminates further production of the exotoxin. Prevention of this disease is achieved by immunizing people with the toxoid during childhood. Booster shots are given approximately every 10 years.

Diphtheria toxin, (DT) secreted by the bacteria Corynebacterium diphtheriae is an exotoxin that functions to terminate host cell functions. DT is composed of two subunits; Subunit A has the catalytic domain which is responsible for inhibiting protein synthesis, and subunit B has the receptor binding region and the translocation region responsible for getting the toxin into the cytoplasm. Subunit B is separated into two distinguished domains, the transmembrane (T) domain and the receptor-binding (R) domain. Alone, subunit A cannot affect cells because it cannot bind to cell surfaces without subunit B.

Binding of the diphtheria toxin to the ganglioside receptor on the plasma membrane causes the molecule to be cleaved into its A and B fragments (Schaechter, 1993). In order for translocation of subunit A into the cytoplasm from the endosome to occur, there must be a conformational change that causes the transmembrane domain to be inserted into the endosomal vesicle. Inside the endosome the disulfide bonds are reduced and this separates the A subunits from the B subunits. Inside the endosomal vesicle, the low pH triggers the translocation of subunit A. Both endocytosis and translocation are necessary for subunit A to reach the cytoplasm. Once subunit A has been successfully translocated from the vesicle, it will then cause its toxic effect in the cytosol.

The mechanism by which cell death occurs is due to a protein modification, ADP-ribosylation. Subunit A targets elongation factor 2 (EF2), which is involved in protein synthesis. The EF2 allows the correct pairing of codons in the mRNA molecule (Alberts, 1994). EF2 has a histidine residue that is highly specific for subunit A exotoxin.

Cell cultures were either bought from biotechnology companies or made in the laboratory. Vero cells overexpressing the receptor sites for the radiolabeled ¹²⁵I-DT were incubated Eagle’s medium. The DT was allowed to bind to the cell’s membrane a time period. The cells were then washed to remove any unbound DT. After internalization of the DT, the cells were treated with Pronase. The Pronase binds to cell membrane cholesterol, makes holes in it, and allows protein leakage. The cell samples were then centrifuged to separate the cytosolic fraction and remnant fraction. See figure below.

The presence of low pH in the endosomal vesicle is required for proper translocation. Tests were conducted in which DT was treated with Bafilomycin A1 to prove that acidification of the intracellular vesicle was needed for translocation of subunit A. Bafilomycin A is an inhibitor of H⁺-ATPase, which inhibits the effects of the DT. Bafilomycin A prevents the translocation of subunit A from the endosomal vesicle to the cytosol. Normally the cell would become acidic, thereby moving subunit A out of the endosome and into the cytosol (Umata and Mekada, 1998).

Nicked and intact DT showed relatively the same amount of toxicity; it’s just that the concentration of them in the cytosol varies. Once the DT has been internalized, cellular proteases, like furin, can cleave intact DT after it has bound to its receptor. The concentration of subunit A’s in the cytosol was three times higher in the nicked DT than in the intact DT (Umata and Mekada, 1998).

Endocytosis of the DT occurs in two major steps, internalization and translocation. DT toxin takes about 20 minutes from the internalization of the DT to the first sign of subunit A being detected within the cytosol. Radiolabeled ¹²⁵I DT was allowed to bind to the plasma membrane for 10 minutes. The cells were then treated with Pronase for 5 minutes to remove excess DT remaining on the cell surface and to prevent anymore of the toxin from being internalized. The amount of subunit A that was translocated was then determined using a BAS 2000 imaging analyzer. The imager detects the radioactivity’s of the fragments. As shown by the data in the Umata paper, subunit A in the cytosol fraction didn’t increase with time, instead after about 6 minutes, there was a decline. What this is saying is that after 20 minutes, if no more new diphtheria toxin is internalized, then translocation of DT from the endosome to the cytosol does not continue. As the supply of DT on the plasma membrane decrease, so does concentration of cytosolic subunit A (Umata and Mekada, 1998).

Studying the effects of how exotoxins are translocated from the outside of the plasma membrane into the cytosol can give some insights as to how other molecules can be introduced into the cell. It is important to figure out how to get other molecules like, growth factors and hormones into the cell so that they can be absorbed or modified by the cytosol. By understanding the mechanism of translocation of the diphtheria toxin, we can relate it to other toxins and their mode of action.

Bibliography

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. 1994. Molecular biology of the cell. 618-621.

Mekada, E., Uchida, T., and Okada, Y. 1979. Modification of the cell surface with neuraminidase increases the sensitivities of cells to diphtheria toxin and Pseudomonas aeruginosa exotoxin. Experimental Cell Research. 123(1): 137-146.

Murray, P., Rosenthal, K. Kobayashi, G., and Pfaller, M. 1998. Medical microbiology. Mosby, St. Louis. 213-216.

Schaechter, M., Medoff, G., and Eisenstein, B. 1993. Mechanisms of microbial disease. 167-168, 181, 295.

Umata, T. and Mekada, E. 1998. Diphtheria toxin translocation across endosome membranes. The Journal of Biological Chemistry. 273(14): 8351-8359.

Copyright © 1999 Sovann Uch and Koni Stone