MADISON, WIS—Our nation’s recent experience with anthrax spread via mail demonstrates only too well the limitations of currently available treatments for advanced stages of this infection. Novel therapies are on the horizon, however. Although these approaches have been tested only in animal models, they may lead to new tools for battling anthrax’s toxic action.

While ciprofloxacin or doxycycline prophylaxis protects people with suspected exposure to anthrax spores, unvaccinated individuals who unwittingly inhale the microbes remain in peril: Following unremarkable flulike symptoms lasting approximately four days, Bacillus anthracis bacilli attain sufficient densities to produce lethal amounts of toxins, triggering a rapid decline in the patient’s condition and, often, death. Because anthrax toxins persist even after antibiotics eliminate the microbes, some promising anti-anthrax strategies target the toxin itself.

TRIPARTITE TOXIN PERSISTS

“Antibiotics stop bacterial growth, but they do not reduce the burden of toxin in the bloodstream,” pointed out John A. T. Young, PhD, Howard M. Temin Professor of Cancer Research at the University of Wisconsin, Madison. Once symptoms recognizable as anything more severe than a common flu develop, it may be too late for antibiotics alone to save a patient. Therefore, he told PULMONARY REVIEWS, “most inhalation anthrax cases treated after symptoms appear lead to death anyway. There is an urgent need to remove this excess toxin.”

Anthrax toxin comprises three proteins: protective antigen (PA), edema factor (EF), an adenylate cyclase that impairs phagocyte function, and lethal factor (LF), a protease deadly to macrophages. Because it acts like a conduit to permit entry of EF and LF into target cells, PA represents a key target for antitoxic strategies.

Leading the invasion of a host cell, PA adheres to the membrane, where a membrane-bound host protease clips it in two. The membrane-bound segment, PA63, is then activated to assemble in groups of seven monomers, forming a heptameric “channel” through which the two other members of the toxic trio, EF and LF, can pass. After binding these toxic enzymes, the PA63 heptamer then enables them to enter the host cell cytosol.

RECOMBINANT-PEPTIDE STRINGS HOBBLE TOXIN

To block PA63’s ability to abet these toxins, R. John Collier, PhD and colleagues sought an agent that could selectively bind the heptameric portal, thereby barring passage of EF and LF across the cell membrane.[1] Dr. Collier, Maude and Lillian Presley Professor of Microbiology and Molecular Genetics at Harvard Medical School, and collaborators first generated a library of bacteriophages, each displaying a random peptide on its surface. Phages bound by a glass surface coated with PA63 heptamers were tested for their ability to compete with EF and LF binding in vitro.

Dr. Collier’s team then chose the peptide from the phage that best bound the heptamer and improved upon it: By gathering an average of 22 copies of this peptide and linking them to a microscopic polyacrylamide chain, they created a potent blocker of LF binding to the PA63 heptamer. At a concentration of only 6 nmol/L, the new agent, dubbed polyvalent inhibitor (PVI), could halve anthrax toxin’s lethality to cells in vitro. Further, twice this dose delayed symptoms in rats co-injected with 10 times the lethal dose of toxin, and a 75-nmol/L dose protected them from toxicity. Thus, the authors conclude, PVI “could be a useful therapeutic ally against clinical anthrax.”

DISABLED PA COMPETES TO BLOCK TRANSLOCATION

In another approach, Dr. Collier and colleagues exploited PA63’s functional requirement for grouping into heptamers: Nonfunctional mutant versions of PA allowed to combine with wild-type PA63 monomers formed “hamstrung” heptamers incapable of translocating EF and LF across the cell membrane.[2] Thus, introducing defective copies of PA could interfere with anthrax toxin’s ability to penetrate host cell membranes. Dr. Collier’s team found one mutant PA that, when mixed 1:1 with wild-type PA in vitro, could assemble with other monomers and completely block toxin translocation. Injected intravenously, a dose yielding a 1:1 or even a 1:4 ratio of mutant:wild-type PA protected rats co-injected with a tenfold lethal dose of anthrax toxin from symptoms. Additionally, the benign yet potently antigenic mutant PA the researchers have developed “may also be useful as a basis for a new vaccine,” Dr. Collier and coauthors suggest.

ZEROING IN ON TOXIN’S TARGET

Another possible approach might be to keep PA from gaining an initial toehold on host cells. Toward this end, a team led by Dr. Young has identified the host cell membrane protein to which PA must bind in order to launch its intrusion. They demonstrate that the anthrax toxin receptor (ATR) is a membrane protein that bears a PA binding site on its extracellular domain.[3] Mutant cells lacking ATR do not bind PA in vitro and are thus resistant to anthrax toxicity, but adding back the gene encoding ATR renders the cells vulnerable to anthrax toxin. Thus, identifying ATR is an important development in understanding how anthrax toxin enters cells and in testing new approaches to disrupt anthrax toxicity.

Further, the researchers identified a specific region of ATR necessary for PA binding and toxin action. The researchers then transformed the portion of ATR containing the pivotal docking site for the toxin into a means of defense: A soluble version of this piece of the protein could bind to PA before the toxin reached its target, thereby preventing its attachment to the cellular receptor.

“The soluble receptor can block toxin action,” Dr. Young noted to PULMONARY REVIEWS. In vitro, 80 nmol/L of this ATR fragment saved 50% of cells exposed to toxin concentrations that are normally more than 90% lethal; at a concentration of 500 nmol/L, the ATR fragment protected 100% of the cells.

“We’re gearing up to do small animal experiments now,” said Dr. Young. “While drugs that perform the same function are being sought, it seems prudent to move forward in animal models with what is an effective inhibitor in culture.” Beside representing a potential anti-toxin therapy itself, the ATR fragment may aid in developing other pharmaceuticals: “This soluble protein can also be used as a new tool for the discovery of small drugs that interfere with toxin–receptor interactions,” Dr. Young remarked.

—Mimi Zucker, PhD

References
1. Mourez M, Kane RS, Mogridge J, et al. Designing a polyvalent inhibitor of anthrax toxin. Nat Biotechnol. 2001;19:958-961.
2. Sellman BR, Mourez M, Collier RJ. Dominant-negative mutants of a toxin subunit: an approach to therapy of anthrax. Science. 2001;292: 695-697.
3. Bradley KA, Mogridge J, Mourez M, et al. Identification of the cellular receptor for anthrax toxin. Nature. 2001;414:225-229.

Return to table of contents