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MICROBIOLOGY From Mutation to Medication? Faulty Toxin Sabotages Anthrax Infection In its airborne form, just a spoonful of Bacillus anthracis, the causative agent of anthrax, could wipe out thousands of people. The speed and ease with which its potent and hardy spores can cause death make the bacterium one of the most dangerous of biological weapons. An estimated 10 countries currently have the capacity to mount an anthrax attack. Its potential as a terrorist tool is equally real. The illustration shows the assembly and entry of anthrax toxin into the cell. Single molecules of the protective antigen (PA) toxin subunit bind to one another to form a doughnut-shaped pre-pore on the cell's surface. The other toxin subunits, lethal factor (LF) and edema factor (EF), bind to the pre-pore, and this complex is endocytosed. In the acidic environment of the endosome, the pre-pore inserts into the membrane and allows LF and EF to enter the cytoplasm, where they wreak havoc on the cell. The pink PA represents a mutant form generated by the authors, which acts as a dominant-negative PA (DN-PA). The presence of a DN-PA in the PA pre-pore prevents the formation of a mature pore, barring entry of LF and EF into the cytoplasm. The blockage of LF and EF inhibits their toxic activities. Illustration by Jeff Cleary Normally, herbivores are the primary victims of the deadly bug, with natural infections of humans rare. A vaccine for humans exists, but the rarity of infection makes mass vaccination impractical. The only post-exposure treatment consists of a standard antibiotic like penicillin, given shortly after exposure. Unless antibiotics are delivered before symptoms develop, the infection progresses rapidly, and unvaccinated victims usually die within a few days. Double-duty Drug Now, researchers at HMS have identified a potential new anthrax therapy. Developed by R. John Collier, the Maude and Lillian Presley professor of microbiology and molecular genetics, and postdocs Bret Sellman and Michael Mourez, the approach may lead to a double-duty drug that acts as an equally effective preventive vaccine and also a more versatile cure. It might be a model for a strategy against other harmful bugs, as well. The study appears in the April 27 Science. B. anthracis secretes the three subunits of its toxin—protective antigen, lethal factor, and edema factor—into the bloodstream of its host. Seven protective-antigen molecules then assemble at the cell surface to form a doughnut-shaped structure to which the enzymatic anthrax toxin moieties, lethal factor and edema factor, bind. The toxin-bound heptameric protective-antigen structure, or pre-pore, is then endocytosed. In the acidic environment of the endosome, the protective-antigen pre-pore undergoes a conformational change that enables it to insert into the surrounding membrane and form a pore. The pore likely provides a passageway for lethal factor and edema factor to translocate across the membrane and enter the cytoplasm. The two moieties primarily enter and destroy macrophages, thereby neutralizing the body's ability to fight off the infection. R. John Collier, Michael Mourez, and Bret Sellman (l to r) developed this new potential anthrax therapy. Photo by Pam Murray Using the crystal structure of the protective-antigen pre-pore, which was solved in collaboration with former HMS researchers Carlo Petosa and Robert Liddington, Collier and colleagues identified amino acids in protective antigen critical for pore formation and translocation of lethal factor and edema factor. Key to future experiments was the discovery that mutant protective-antigen molecules were still capable of binding to wild-type protective-antigen molecules and to lethal factor and edema factor. This finding led the researchers to hypothesize that these mutants might, in dominant-negative fashion, interfere with mature pore formation and prevent toxicity, thereby serving as a therapy for anthrax infection. Furthermore, they reasoned, this treatment might be effective after traditional antibiotics have stopped working. The Mutant Effect The researchers first tested the ability of protective-antigen mutants to prevent wild-type protective-antigen–dependent translocation in vitro. Four of the six mutants they tested were effective, the most potent of which was almost completely inhibitory at a ratio of 1:1 with wild-type protective antigen. This suggested that a single mutant in an otherwise wild-type heptamer corrupts the entire complex. The researchers then assessed the ability of the protective-antigen mutants to impair toxin action in the classical in vivo anthrax model, the rat. When protective-antigen mutants were injected together with a normally lethal mix of wild-type protective antigen and lethal factor, the rats did not develop any symptoms of intoxication. The protective-antigen variants "totally protected the animals, whereas the control animals became moribund within 90 minutes," said Collier. The three most potent protective-antigen variants fully prevented symptoms when present in a 0.25:1 ratio with wild-type protective antigen. "The results are remarkable," Collier said. Variants of protective antigen are particularly good candidates for an anthrax therapy because wild-type protective antigen is already known to be safe in humans: it is the major component of the current anthrax vaccine, the use that gave the subunit its name. Demands of Prevention Mutant protective antigen also elicits an immune response equivalent to that generated by wild type in rats. Mutant protective antigen, therefore, may fill the bill as both an anthrax vaccine and treatment, negating the need to produce two separate drugs. Mutant protective antigen may also prove to be important in preventing delayed anthrax, the situation in which anthrax develops weeks after exposure due to delayed spore germination. "The hope is that by administering this molecule you would elicit an immune response that would have generated protective levels of antibodies against the toxin by the time these spores germinated," said Collier. The researchers are now determining whether more potent inhibitor protective-antigen molecules can be made. Future efficacy studies using a mouse model of anthrax infection will be performed at U.S. Army labs in Maryland. If shown to be effective later in the infection cycle than current antibiotics, these mutants may be developed into an anthrax treatment. To wield their tools of destruction, certain other disease-causing bacteria, such as staphylococci, also require the formation of structures similar to the protective-antigen pore. Therefore, "it may be possible to generalize this approach to certain other diseases," Collier said. With the rise of antibiotic-resistant bacteria and the threat of biological warfare, new therapies such as this are demanding attention. —Heather Ettinger