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Receptor-Based Strategy Neutralizes Diphtheria Toxin, Maybe Others

A potential new means for combating diphtheria, a deadly disease for which vaccine-based preventive measures ordinarily work effectively, could also prove applicable to other toxin-based infectious diseases that are worrying public health officials these days. Moreover, basic insights into mammalian cell receptors to which such toxins bind could lead researchers to develop means for exploiting the biological ingenuity of pathogens as a way to defend against the diseases those pathogens can cause.

In the May issue of Infection and Immunity (70:2344-2350), Leon Eidels and his collaborators of the University of Texas Southwestern Medical Center in Dallas report that a piece of the diphtheria toxin receptor's extracellular domain can prevent the full toxin from binding to target mammalian cells. Entry of that toxin into mammalian cells is the key step that leads to the devastating symptoms associated with infections caused by Corynebacterium diphtheriae, including fever, localized pain, and obstruction of the airways. This disease, long in check due to wide use of effective vaccines, reemerged in countries of the former Soviet Union during the 1990s when their public health systems were in shambles. Other countries also saw reemergence of this deadly disease, underscoring the need for a better treatment than equine diphtheria antitoxin, which is effective in emergencies but is in relatively short supply and can have nasty side effects.

"When we began looking for it, nobody knew what the receptor for diphtheria toxin was," says Eidels, alluding to his long-term quest that led to his isolating the diphtheria toxin receptor 10 years ago. "All they knew is that possibly it's a protein, because when they treat cells with proteases they became less sensitive." He also knew that monkey Vero cells are sensitive to diphtheria toxin, whereas Vero cells from mice ordinarily are not. Hence, he transfected mouse cells, known to be resistant to the toxin, with genetic material in a cDNA library derived from toxin-sensitive monkey cells. "We then had to screen about 10,000 colonies in order to find one that became sensitive to the toxin," he says. "The rest were still resistant!"

When Eidels and his collaborators sequenced the cDNA that rendered the mouse cells sensitive to that toxin, they were surprised with what they found. "The sequence was that of the membrane-anchored form of the precursor of Heparin-binding EGF-like growth factor," he says. "The fact that it was a growth factor was a big surprisebecause no other growth factors seemed to be receptors for toxins.

"Once we realized this was a precursor to a growth factor, an important protein, we realized mouse cells must have it, but it must be in a different form," Eidels says. "We cloned the mouse equivalent, sequenced it, and found that it is very similar, except in a couple of regions, which suggested that those might be binding regions. We narrowed it down to one region, and we were able to identify exactly where the toxin binds. It's a region where mouse and monkey differ a lot."

The growth factor itself seemed a good candidate for neutralizing free toxin, Eidels says. However, because growth factors can also be tumorogenic, he adds, "We changed it in such a way that it is no longer tumorogenic, but it still binds the toxin, and so would be a good candidate for an antidote." According to in vitro studies, "it was a more effective inhibitor of diphtheria toxin binding than the recombinant mature heparin-binding EGF," he says, suggesting that it could be an effective antidote for ongoing cases of human diphtheria. Studies are planned to determine whether it can protect toxin-sensitive transgenic mice against the toxin.

Diphtheria was among the first infectious diseases for which treatment became available, in 1898, says James Caper of the University of Maryland Medical School in Baltimore. However, that treatment, consisting of antibodies from the blood of immunized horses, causes serum sickness in about 10% of cases. "Now we have a real molecular approach to blocking toxins with these receptor analogs," he says. "Hopefully, it can block the binding of the toxin without causing ill effects to the host." This would be the first new treatment for this disease in more than 100 years.

Centers For Disease Control and Prevention

But Alison O'Brien of the Uniformed Services University of Health Sciences in Bethesda, Md., and editor in chief of Infection and Immunity, doubts that the new diphtheria toxin-blocking compound will be developed commercially anytime soon. "It's expensive, and the countries that would spend the money to do it are the ones that have a full vaccine regime in place," she says. However, the threat of terrorism underscores the importance of developing antidotes to diphtheria and other toxins, points out Joseph Barbieri of the Medical College of Wisconsin in Milwaukee. The strategy being developed by Eidels and his collaborators "would be applicable to most toxins that have high-affinity receptors," he adds. The Centers For Disease Control and Prevention lists several microbiologically derived toxins on its website describing bioterrorism threats that might fall into this category.

Not only is a toxin receptor-based approach promising for treating diphtheria, but "this approach could be useful for other toxin-mediated diseases," Eidels says. For example, John A. T. Young of the University of Wisconsin in Madison and R. John Collier of Harvard Medical School in Boston, Mass., are pursuing a receptor-based approach to neutralizing the anthrax toxin. They recently identified the mammalian receptor for this toxin, and call it anthrax toxin receptor (ATR). Noting that no one yet knows its normal function, Eidels wryly notes, "God in her wisdom did not put this protein on the cell surface to be a receptor for a toxin to kill the cell." In any case, Young and Collier are using information about the receptor's molecular structure to develop and test potential inhibitors of anthrax toxin activity, including a compound called sATR, which is a soluble form of the receptor domain.

Glen Armstrong of the University of Alberta, Edmonton, is applying this same approach to developing an antidote for shigatoxin, produced by Escherichia coli O157. "We're getting good protection in a mouse model," he says.

David Holzman
David Holzman writes from Lexington, Mass.