Scientists Getting to the Core of Bacillus anthracis

Proteins have a notoriously difficult time traversing the hydrophobic layers of the plasma membrane. But some species, such as Bacillus anthracis, have devised clever ways to push their proteins through. Anthrax kills with a toxin, a compound composed of three proteins—protective antigen (PA), lethal factor (LF), and edema factor (EF)—that somehow penetrate the plasma membrane of the host cell and enter the cytosol where they make their kill. Antibiotics against anthrax attack the bacteria, not the deadly toxins that the bacteria secrete. If a patient does not receive antibiotics in the infection's early stage, he or she can die from accumulated toxin. But if antitoxin drugs were developed, they could be administered at this later stage of illness, and they could save lives.1 For years, scientists have been quietly working behind the scenes, trying to understand how anthrax toxins can penetrate the hydrophobic layers. While researchers do not have all the answers, they do have some workable theories. John Collier, a researcher at Harvard Medical School, and his colleagues started investigating anthrax toxin inhibitors as a way "to help us dissect out its fundamental mechanism and mode of action," he says. Though Collier's original ambitions were purely scientific, "when something becomes obvious, when you come across a finding that has practical uses, certainly we follow that line of research." Collier's studies have led to several potential antitoxin therapies to treat anthrax. Last year, Collier and two of his postdocs, Michael Mourez and Bret Sellman, described a mutant form of PA known as dominant negative inhibitor (DNI), which completely blocks toxin action.2 Later that year, in collaboration with George Whitesides of Harvard, the re-searchers coauthored another paper describing a second type of anthrax antitoxin, the polyvalent inhibitor.3 And in November, in collaboration with scientists from MacArdle Laboratory for Cancer Research at the University of Wisconsin, Madison, Collier's team described a third potential antitoxin strategy based on soluble forms of the anthrax toxin receptor, the site on the surface of the host cell where the anthrax toxin binds.4 "These are all interesting approaches," says Sjur Olsnes of the Norwegian Radium Hospital, Norway, but "it is hard to say which one will make it into practical use." Why PA? All three inhibitors target only PA, but the other two toxin subunits, LF and EF, actually do the damage; importantly, neither is toxic by itself. The only way LF and EF can get across the plasma membrane and into the cell is by forming complexes with donut-shaped rings of PA molecules that have already bound to the cell surface. Because "PA is the part that binds to cells, if you can prevent PA from acting then you prevent the action of LF and EF at the same time," says Mourez. After PA, LF would probably be the next best bet for an antitoxin strategy because "LF is what kills," says Mourez. "We don't know how it kills, but it does." Andrew Bohm of the Boston Biomedical Research Institute in Watertown, Mass., agrees, "EF is the least sensible of the three toxins to be targeting. LF, after all, is the one that kills. EF plays only an ancillary role; it causes edema, but it doesn't cause septic shock." Nonetheless, until antitoxin drugs are developed and tested, it makes little sense to target any one of the toxin subunits. Regardless of the target, says Bohm, the goal should be to develop drugs with the fewest side effects when administered in combination with antibiotics. The Antitoxins DNIs are inactive PA proteins that can block the toxicity of the entire PA-LF or PA-EF complex by preventing it from crossing the plasma membrane. Collier and his postdocs identified mutant PAs that are so potent, Collier says, that only a single molecule need be incorporated into the PA heptamer to stop the toxin in its tracks. They injected mixtures of PA's mutant and lethal forms into rats and found that the mutant structure showed no signs of toxicity. Rats that did not receive the mutant form died within 90 minutes. The next step, says Collier, is to test DNIs in infected animals, but they cannot do this at Harvard because of the specialized containment facilities required. "This is a real problem right now," says Collier, who has been negotiating with the US Army so that the testing can move forward. But, he says, the Army has not been able to provide the resources. The DNIs "seem to be the most promising" of the approaches, Collier says. "What it will take to get FDA [Food and Drug Administration] approval is not entirely clear," but normal (not mutant) PA is already known to be safe in humans. Indeed, PA is the major component of the current anthrax vaccine. DNIs not only stop the toxin, but they have the added advantage of eliciting an immune response, thereby boosting the immune system. Polyvalent inhibitors (PVIs), in contrast, do not have this capability. Soluble versions of another antitoxin, anthrax toxin receptor, have been tested in cell cultures and are the farthest from the clinical trial stage. Whereas DNIs prevent the assembled toxin from moving across the membrane, PVIs inhibit the assembly process itself by preventing LF and EF from binding to the PA heptamer. In collaboration with Whitesides, Collier's team constructed the anthrax PVI by linking 22 copies of an inhibitory peptide on a flexible, molecular, Velcro-like polyacrylimide backbone. By themselves, the peptides that comprise a PVI have a very weak affinity for the heptamer, says Mourez. But when they are linked together in a way that exposes many peptides simultaneously to the heptamer, there is as much as a 10,000-fold increase in activity on a per-peptide basis, says Collier. In the past, Mourez explains, researchers had to know exactly where a peptide binds for this polyvalent approach to work, so they experimented with rigid backbones that relied on very precise molecular modeling. But Mourez and his colleagues took a different approach. Even though they did not (and still do not) know exactly where the inhibitory peptide binds, the flexibility of the polyacrylimide backbone increases the chance that at least one of the peptide copies will find the binding site. That it worked was a surprise, says Mourez. Whitesides had experimented with these flexible backbones before, but this was the first time they had ever been tested in an animal model. Rats normally die within hours after exposure to anthrax toxin, but rats that were injected with PVIs showed no signs of toxicity. As with the DNIs, the next step will be testing the efficacy of PVIs in an infectious setting, says Mourez. His group has already tested the effect of PVIs on toxicity in rats, but "we really don't know much about how the toxin is killing people and how it kills in the context of infection." What's Next? "The anthrax inhibitor is very peculiar," says Mourez, because its subunits assemble on the outside of the host cell. Most multisubunit bacterial toxins are preassembled inside the bacteria before they are secreted. Scientists hope that the anthrax inhibitors will serve as useful models for fighting infections caused by other toxins with similar cell-surface assembly lines. For example, the C2 toxin secreted by Clostridium botulinum, the bacterium that causes botulism, also assembles on the host cell surface and could potentially be blocked by a dominant-negative mutant, as could the VacA toxin secreted by Helicobacter pylori, the bacterium that causes gastric ulcers.5 In light of last year's deaths from inhalational anthrax and the heightened awareness of bioterrorism, the inhibitors that Collier and his collaborators are studying have attracted considerable attention, which, says Collier, has been good and bad. It "was a distraction for a couple months," he says. But "other people come out of the woodwork once they realize what you are doing, which has resulted in some fruitful collaborations." The scientists are using facilities at Harvard for further computational screening of small molecules that may be useful as inhibitors, says Collier. Harvard is in the process of negotiating the licensing of some of the technology. Leslie Pray (lpray@nasw.org)is a freelance writer in Leverett, Mass. References 1. L. Pray, "The B. anthracis picture is now complete," The Scientist, 16[4]:28, Feb. 18, 2002. 2. B.R. Sellman et al., "Dominant-negative mutants of a toxin subunit: An approach to therapy of anthrax," Science, 292:695-7, 2001. 3. M. Mourez et al., "Designing a polyvalent inhibitor of anthrax toxin," Nature Biotechnology, 19:958-61, Oct. 1, 2001. 4. K.A. Bradley et al., "Identification of the cellular receptor for anthrax toxin," Nature, 414:225-9, Nov. 8, 2001. 5. S. Olsnes, J. Wesche, "Fighting anthrax with a mutant toxin," Science, 292:647-8, April 27, 2001.

Scientists Getting to the Core of Bacillus anthracis

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