Science Editor

The U.S. military has enlisted molecular biologists at Rockefeller University in New York to fight the anthrax pathogen (Bacillus anthracis) on its own turf. The scientists in turn have recruited the oldest and most abundant form of life on earth to wipe out millions of anthrax bacteria within seconds.

This killer virus is the bacteriophage (literally "bacteria-eater") that infects B. anthracis. The pathogen makes use of its phage to escape from an anthrax-infected host - whether man or beast. That specific bacterial virus deploys an anthrax-killing enzyme named PlyG lysin.

"That's a protein, an enzyme," explained Rockefeller's Raymond Schuch, a molecular biologist in the Laboratory of Bacterial Pathogenesis and Immunology, headed by Vincent Fischetti. "Normally," Schuch noted, "the viruses are growing inside the bacterium. They reach a certain density, then open up little holes in the bacterial membrane and insert the PlyG lysin protein into its cell wall. It punches lytic holes that immediately destroy the bacterium."

Schuch is first author of a report in last week's Nature, dated Aug. 22, 2002. It's title: "A bacteriolytic agent that detects and kills Bacillus anthracis." Fischetti is the paper's senior author.

"We now know that anthrax is a real threat," Fischetti observed. "But even more of a threat are multidrug-resistant strains of anthrax, which may occur naturally or be engineered by terrorists using common molecular techniques. Consequently, alternative strategies for combating these dangerous strains are needed more than ever." Fischetti and his team have turned PlyG lysin into a powerful new agent that, besides instantly laying low anthrax bacteria, shows promise as a means of detecting the pathogen and its deadly spores.

"The finding of our Nature paper identified a novel antibacterial enzyme, an antibiotic that can kill Bacillus anthracis," Shuch told BioWorld Today. "And we've exploited this finding in order to demonstrate the therapeutic and diagnostic potentials of PlyG lysin. Its novelty lies in the source of our agent, which is a bacteriophage, whereas existing antibiotics normally come from bacterial or fungal soil organisms. Ours is completely different. It's a bacterial virus, from which we have identified this lysin product."

Unlike Most Antibiotics, Bug Puts Up No Resistance

"As far as its novelty goes," he continued, " the target of our enzyme - an amide bond in the surface of the bacterium - has not been exploited as a target by other antibiotics. Its interesting feature is that unlike conventional antibiotics, we do not get any resistance to ours. That is, the target cannot be mutated by the bacterium to get around the action of our enzyme."

Funding this research is DARPA - Defense Advanced Research Projects Agency, the U.S. Defense Department's central R&D organization. "We got a grant for about $2 million from DARPA," Shuch said, "so naturally we began work on Bacillus anthracis. The military is primarily interested in it as a therapeutic agent," he added. "They have to get FDA approval first for PlyG's use, and that involves showing efficacy and nontoxicity to humans in two animal models of infection - rabbits and eventually monkeys. We'll get a contract lab to do that for us.

"I think we can begin within six months or so," Shuch went on. "We'll have sufficient enzyme produced for us by then. Once that's done, if everything is OK, the military, which is paying for this, can begin to stockpile what it envisions as an injectable therapeutic agent to give people infected in the event of an anthrax attack. It would probably be administered in conjunction with another antibiotic and an antitoxin. I think the length of time envisioned for that to happen is two or three years.

"Spore detection," Shuch pointed out, "is probably a lot closer to being used. We've performed many of the initial experiments already in our laboratory. We're just waiting again for sufficient enzyme, so that we can try some field tests showing natural detection of these anthrax spores. Then it'll be ready."

What he calls "the spore-detection machine" starts by capturing the bacterial organisms on a filter. To this is added a spore germinant. If any spores are present, the germinant will break their dormancy so they start growing. The lysin splits them open and releases ATP - adenosine triphosphate - from the spores' interior. Firefly luciferase reflects ATP's glow, which can be detected by a hand-held luminometer.

"That's the basic framework," Shuch pointed out. "It's fairly rapid and very portable. You can actually go to a site of suspected spore contamination and test right there in situ, rather than having to send samples to some clinical laboratory." He made the point, "We'll have to produce enough enzyme to do this. Probably within a couple of months the luminometer can be ready to go."

Mice Now, Rabbits, Monkeys Later

At present, Shuch and his co-authors are testing their phage protein on mice. "Once we had isolated the enzyme," he recounted, "we showed that our Bacillus cereus - a look-alike to anthracis - is fatal when injected into the mouse's peritoneal cavity. We gave them a dose of cereus such that 100 percent of the mice died within five hours after getting the pathogen alone. We then hit them with the enzyme 15 minutes post-infection with the bacteria. Roughly 75 [percent] to 80 percent of the mice were rescued, surviving at least four or five days afterward and appearing completely healthy and normal.

"The next step was performed with anthrax spore infection in mice, and those are ongoing right now. We expect preliminary results in the next month or two."

Shuch said, "We're currently working on two other biowarfare agents. One is Yersinia pestis, the cause of bubonic plague; the other, Vibrio cholerae. But we have plans to extend this to other weaponizable pathogens in the near future, beginning with tularemia. That devastating disease, caused by Francisella tularensis, is transmitted to humans from rodents through the bite of a deer fly."

Shuch summed up: "I think the interesting thing here is our exploitation of the bacteriophage, and looking to them for sources of other antibacterial agents. Phages are the most abundant organism on earth. They've been killing bacteria for billions of years, and are pretty much unexploited with respect to developing antibacterials," he concluded, "and we're starting to do that."