The anthrax toxin is believed to play a critical role in causing disease symptoms, in many cases leading to death even when antibiotics have been administered to stop bacterial growth. Consequently, there is a great deal of interest in better understanding how the toxin functions so that effective inhibitors with known mechanisms of action can be developed.
The findings, published in the online early edition of the Proceedings of the National Academy of Sciences, come from a joint study by scientists at the Salk Institute for Biological Studies and Harvard Medical School.
"This research tells us that we need to revise the model of anthrax toxin entry so that more effective drugs can be developed," says the study's principal investigator, John A. T. Young, Ph.D., a professor in the Infectious Disease Laboratory at Salk. In previous research on the mechanisms used by the anthrax toxin to invade cells, Young's group discovered that the toxin targets two different types of receptors known as TEM8 and CMG2, on the cell's surface.
In the new study, Young and his colleagues found that toxin entry occurred at near neutral pH conditions when it was bound to the TEM8 receptor, but at strongly acidic conditions when bound to CMG2. The researchers in Young's lab at Salk revealed the two different pH levels when they created cells that had only TEM8 receptors or CMG2 receptors, but not both. They then used a drug that neutralizes the pH inside cells to test how it affected toxin entry in both cell types.
To their great surprise, the toxin behaved differently in the two cell types. For cells with the TEM8 receptor, a near-neutral pH was sufficient for toxin entry, but the cells with CMG2 required much more acidic conditions. Although the types of receptors found on different cells in the body have not yet been well defined, the investigators went on to show that different cultured cell lines display either of these two behaviors.
"Pore formation and translocation of the toxin occurred under strikingly different conditions," Young says. "The finding that receptor type dictates different pH thresholds was completely unexpected."
The lack of a uniform pH threshold suggests that the anthrax toxin may take two alternative pathways to reach different regions inside the cell, and that "drugs that target a single pathway may be ineffective," says Jonah Rainey, Ph.D., a research associate in Young's lab and the lead author of the PNAS paper.
Pharmaceutical agents that are designed to block the acid-dependent route of toxin entry may fail to block the neutral pH-dependent pathway, the researchers say.
In addition, the researchers made a finding that could potentially lead to a new approach for blocking toxin entry into a cell. The toxin forms a syringe-like pore through which the active parts of the toxin can enter the cell.
The scientists found that pore formation is associated with release of the receptor from the toxin. "Prior to this work it was thought that the receptor was only partially released during toxin pore formation but our results suggest that that complete receptor release is required," says Rainey
"This is a key new finding, because blocking receptor release might disarm the toxin," says Young.
Rainey adds, "A pharmaceutical agent that keeps the receptors locked in their original position may render the anthrax toxin harmless."
Anthrax is an acute infectious disease caused by the spore-forming bacterium Bacillus anthracis. It is most commonly found in such animals as cattle, sheep and goats, but it can also occur in humans as a result of exposure to infected animals or to anthrax spores used as a bioterrorist weapon, as was the case in 2001 when letters containing spores were mailed through the postal system.
Co-authors on the study, which was funded by the National Institutes of Health, include Salk researchers Patricia L. Ryan and Heather Scobie, and Darran J. Wigelsworth, Ph.D., and John Collier, Ph.D. of Harvard Medical School.
The Salk Institute for Biological Studies in La Jolla, California, is an independent nonprofit organization dedicated to fundamental discoveries in the life sciences, the improvement of human health and the training of future generations of researchers. Jonas Salk, M.D., whose polio vaccine all but eradicated the crippling disease poliomyelitis in 1955, founded the Institute in 1960 on land donated by the City of San Diego and with the financial support of the March of Dimes. For more information: www.salk.edu.