Science 1663

To Kill a Killer—Targeting Anthrax

Los Alamos researchers work to counter a bioterrorism threat

The rod-shaped bacterium, Bacillus anthracis (magnified 2,200 times), causes anthrax. Its spores (shown forming at the center of the image)—an inert, protected stage—allow it to survive hostile environments until it invades a new victim. IMAGE courtesy of Dennis Kunkel Microscopy, Inc

Autumn 2001 was a time of terror, some of it arriving in the mail. A still-unknown someone sent envelopes of anthrax spores (dormant cells, ready to come to life) to several news organizations and to the Washington, D.C., offices of two U.S. senators.

There were only seven envelopes, but the consequences were enormous. Twenty-two office workers and mail handlers contracted anthrax. Five died. Bags of mail were impounded, thousands of people were given precautionary medical treatment, and dozens of contaminated buildings were temporarily closed, one for 4 years.

Those few envelopes caused grief, fear, and a multi-million-dollar bill for response and cleanup. They also put us on alert. Naturally contracted anthrax is extremely rare in humans, but a deliberate biological attack could expose a large population and massively disrupt the economy.

We may never know the enemy behind the 2001 mailings, but the enemy we can know is the bacterium that causes anthrax, Bacillus anthracis (or simply B. anthracis). The better we know it, the better the chance of averting disaster.

Los Alamos Weighs In

Right from the beginning, Los Alamos National Laboratory has been a key participant in the fight against anthrax. It contributed to the DNA forensics used by the Centers for Disease Control and Prevention to identify the 2001 strain of B. anthracis (the Ames strain, the so-called "gold standard" for virulence). The Laboratory's longstanding expertise in DNA sequencing has made it a significant player in DNA forensics, wherein patterns in DNA are probed for information about an organism's provenance.

Sailors aboard the aircraft carrier USS John C. Stennis receive the first in a series of vaccinations for anthrax. The vaccine, available for military personnel and first responders, requires multiple shots and takes many months to confer immunity.Photo courtesy of U.S. Department of Defense

Currently, Los Alamos is expanding its research toward better understanding of the life cycle of this deadly bacterium. It is also developing new medical treatments to supplement current ones, which have serious limitations. The current vaccine induces immunity through the production of antibodies but requires months of vaccinations and an annual booster. It would be of little help in an emergency, like a bioterrorist attack. In addition, bacterial mutations—natural or, in the case of bioterrorism, deliberate—can reduce the vaccine's effectiveness.

Similarly, weaknesses undermine the antibiotics used to treat anthrax victims. The most-prescribed antibiotics cause side effects because they're "broad spectrum," attacking many types of bacteria at once, including the beneficial bacteria in the gastrointestinal tract. And mutated bacteria may be resistant to antibiotics. To defeat something like B. anthracis, you start with how it overwhelms the body's defenses and look for vulnerabilities in what is a seriously tough customer.

A Formidable Opponent

Outside a living host, B. anthracis forms spores that can resist heat, dehydration, and even radiation and can survive for years, long enough to invade a new host. The spores can be ingested with infected meat or enter the body through a break in the skin. They are so tiny (a micrometer—one 25-thousandth of an inch) they can disperse through the air and be inhaled, a potent path to infection attractive to bioterrorists.

Inhaled spores, magnified here 1,795 times, are trapped in the smallest of the lungs' air sacs. Dennis Kunkel Microscopy, Inc

Inhalation anthrax has a mortality rate of 50 to 75 percent if not treated quickly, but it's hard to catch quickly because its early symptoms are like those of a cold or the flu. The 2001 victims who died were the ones who inhaled the spores.

Inhalation is especially dangerous because it places the spores deep within the respiratory system, beyond all the body's outer defenses, and on a quick pathway to system-wide infection.

The spores lodge in the lungs' tiniest air sacs, the alveoli. There they encounter mature white blood cells called macrophages (Greek for "big eaters"). Those should be the spores' undoing. Macrophages normally envelop and break up (eat) invading pathogens, then travel from the alveoli to the lymph nodes, displaying bits of the pathogens on their outer surface. Those displayed bits signal the white blood cells that reside in the lymph nodes to produce antibodies.

Although the anthrax spores are enveloped as usual, they co-opt the macrophages for their own purposes. They resist the "eating" and instead germinate into fully active B. anthracis bacteria, which ride along, undamaged, to the lymph nodes. There they burst out to multiply at a staggering rate—and secrete a deadly toxin—in the favorable environment of the bloodstream.

Breaking and Entering - Anthrax Toxin Invades a Cell. The anthrax toxin comprises three proteins (PA, EF, and LF) that work together to break through the cell's outer wall and set off a string of reactions that can ultimately kill the host.

The toxin is a complex of three proteins working cooperatively to kill cells. The first one, called protective antigen (PA), initiates the attack. It binds to a receptor found on almost every cell in the body and lets itself be pulled inside, dragging along the other two proteins—edema factor (EF) and lethal factor (LF). EF and LF can't enter a cell on their own and are harmless outside. Inside, they set off a string of death-dealing events, including the malfunction and swelling of cells and disruption of the intercellular signals that would activate the immune system.

Launching a Decoy

Momchilo Vuyisich, co-developer of the Los Alamos antitoxin.

PA is the focus of an antitoxin being developed by a Los Alamos team of Goutam Gupta (heading the team), Momchilo Vuyisich, and "Gnana" Gnanakaran. Team members want to block the toxin by keeping PA from attaching to a cell's receptors.

The antitoxin is a molecular decoy shaped like a human antibody (a Y-shaped protein that helps the body clear invading organisms). The branches of the decoy's Y contain replicas of the cell's PA receptors. PA binds to the replicas instead (see illustration). The decoy's tail is identical to that of a human antibody, and its presence prompts the immune system to clear the decoy and its captured toxin from the body.

The current anthrax vaccine functions similarly—but not as well. It induces the body to produce its own anthrax antibodies, but over a period of months, not immediately. Those likewise bind to PA, but the Los Alamos decoy binds to significantly more area on PA. To escape the decoy, PA would have to accumulate multiple mutations over a wide area. In contrast, a single mutation could allow it to escape the grasp of the vaccine-produced antibody.

In addition, the decoy's antibody tail gives it stability because the immune system sees it essentially as "one of its own." Like a normal human antibody, the decoy is allowed to exist unhindered until needed.

The decoy has passed several major tests sponsored by the National Institute of Allergy and Infectious Diseases and by the National Institutes of Health, Biodefense Program. The tests were carried out on rats and mice at the University of New Mexico's Health Sciences Center. "In about 18 months, if all goes well, we should be ready for trials using infected human blood." Gupta says. "That would bring us more or less to human trials."

Metabolism and Mystery Proteins

Other Los Alamos researchers are pursuing the anthrax bacterium down different paths.

A team headed by Christy Ruggiero and Andy Koppisch is trying to develop new antibiotics that specifically target B. anthracis through its metabolism. The idea is to block the bacterium's acquisition of iron. Most bacteria require dietary iron to function, and they use a special molecule—a siderophore ("iron carrier")—to bring it in from the environment. The siderophore used by anthrax is called petrobactin. The team's goal is to keep B. anthracis from taking up that iron-loaded molecule, effectively starving the bacterium.

The figure shows how the Los Alamos antitoxin, a Y-shaped molecular decoy (yellow, violet, and green), binds two PA molecules (red). PA is the toxin component that binds to human cells. Both the decoy and PA are proteins, long chains of amino acids, depicted here as folded ribbons. The binding configuration shown was calculated by a computer simulation. The inset shows the molecular components of the decoy: the PA receptor (yellow), the human antibody tail (green) and the linker between them (violet).

"As far as we know," says Ruggerio, "Bacillus anthracis is the only pathogen that uses petrobactin."

Currently, the team is identifying and studying the effect of turning off several of the bacterium's genes that control petrobactin uptake. Once the critical genes are identified and the proteins those genes express (produce) are examined, team members will work on antibiotics that block those proteins.

Other Los Alamos teams are pursuing other metabolic vulnerabilities.

Koppish is leading a separate team that is looking into other metabolic functions and investigating how the bacterium develops resistance to antibiotics, in the hope of preventing resistance before it happens.

Because the bacterium's metabolism depends on enzymes, researcher Paul Langan's team is channeling its anti-anthrax work into an attack on one particular enzyme, DHFR, which is needed for the bacterium's synthesis of DNA and therefore essential for its survival.

Cliff and Pat Unkefer are working in a new field of study known as "metabolomics," named for the molecules called metabolites that are its focus. Metabolites are the products of metabolism. They offer a window into the biochemical workings of an organism. The Unkefers' recent work with the metabolites in B. anthracis has given them leads to new targets for drugs.

Ryszard Michalczyk's anti-anthrax team is studying six extra genes unexpectedly found on a circular piece of B. anthracis DNA—a plasmid, the same one that also contains the genes expressing the three toxin proteins. The extra genes also express proteins but of unknown function. Their proximity to the toxin suggests an association, which if it exists, would make them additional drug targets. Michalczyk is hoping so.

Joining Forces

Sometime in the future, a combination of these Los Alamos strategies may work in tandem. For example, an antibiotic interfering with B. anthracis's metabolism might slow the bacterium's growth and thereby reduce the amount of toxin that the antitoxin decoy needs to block. And all of them may supplement the vaccine, extending its efficacy.

Separately and together, all research avenues may lead to a future that is safer from bioterrorist attack.

• Biosecurity & Public Health
• CDC Antrhax
• Mayo Clinic Anthrax