By David N. Leff

Is anthrax old hat? Is the Ebola virus too hot to handle? So what's new in biological warfare?

A half-page article in Tuesday's New York Times carries the headline: "Iraq Suspected of Secret Germ War Effort." In this piece, arms experts suggest that among the pathogens Saddam Hussein may be clandestinely stockpiling are "the organism causing bubonic plague [Yersinia pestis], aflatoxin, a fungal agent that can lead to liver cancer," plus "botulinum toxin, anthrax, camel pox and infectious hemorrhagic conjunctivitis virus."

How come that list leaves out Mycobacterium tuberculosis - a germ warfare natural? A couple of billion people - approximately one-third of the world's population - are infected with M. tuberculosis (TB). It's the leading cause of death from a single infectious agent, killing 3 million victims a year. And TB is the main perpetrator of mortality among AIDS casualties.

The TB bacterium seem so tailor-made to be a biowar agent because of the cunning way it spreads its infection. What usually starts with a cough, fever, sweating, night sweats and shortness of breath ends up with debilitating - frequently fatal - bacterial residence in the lungs. It's the cough - or perhaps a kiss - that passes the microdroplets of TB contagion to its next victim.

It didn't take a modern dictator to launch - or rather re-launch - this age-old malady. M. tuberculosis has been a fellow traveler with Homo sapiens since man took up farming 10,000 years ago, and acquired the bug from his cattle. The disease waxed and waned throughout human history, but seemed terminally vanquished half a century ago with the discovery of an antibiotic called isoniazid. This TB-specific drug quickly emptied tuberculosis sanatoriums, and people began regarding M. tuberculosis as a has-been germ.

"Isoniazid is a drug that works specifically by inhibiting the biosynthesis of the bacterium's cell wall," said structural biochemist Donald Ronning, at Texas A&M University in College Station. "It inhibits the synthesis of mycolic fatty acids that incorporate the major portion of that unusual cell wall." But ironically - or inevitably - isoniazid, the first and foremost TB antibiotic, became the first target of the pathogen's drug-resistance backlash. (See BioWorld Today, Oct. 27, 1999, p. 1.)

How Mycobacterium Blackballs Isoniazid

"One of the ways that mycobacteria are resistant to traditional antibiotics," Ronning explained, "is that they produce beta-lactamase enzymes, which are generally found in the space between that outer cell wall and the bacterium's inner cell membrane. So any traditional antibiotic that penetrates this thick wall gets chewed up by the beta-lactamases, and is no longer functional as an antibiotic.

"Also," he added, "it's thought that the cell wall acts as a barrier against diffusion of the antibiotic into the cell to begin with. So very little of it gets inside the bacterium."

But besides the bug's drug-resistance strategy, TB has other powerful culprits - its own human society. An editorial headed "Taming tuberculosis - again" in the February 2000 issue of Nature Structural Biology lists the five main "interrelated factors" that caused the number of cases in the U.S. and around the world to resurge in the mid-1980s: the HIV/AIDS epidemic; increased immigration from countries with many TB cases; increased poverty and homelessness; decline in the health-care infrastructure; and poor patient compliance with treatment regimens.

The bottom line of this rap sheet is the urgent need for new antibiotics and vaccines to beat back the current M. tuberculosis onslaught. This in turn calls for research expeditions into the heart of the bacterium's darkness - namely, its fine-grained atomic structure.

The latest chart derived from such exploration appears in that same February issue of Nature Structural Biology, titled: "Crystal structure of the secreted form of antigen 85C reveals potential targets for mycobacterial drugs and vaccines." Ronning is the report's lead author.

Why 85C? "This family of the antigen 85 proteins," Ronning told BioWorld Today, "has been shown for about 30 years now to elicit an immune response in humans.

"Then a couple of years ago," he went on, "it was determined that this 85 protein is responsible for much of the biosynthesis in that cell wall. So now we're looking at these enzymes as a model for designing a new vaccine against TB, but also in the short term so that we'd be able to design a new family of antibiotics that would block these proteins."

Antigen 85 comes in three nearly - but not quite - identically homologous versions - A, B and C.

"We just happened to pick C to work on," Ronning recalled. He's now turning to the X-ray crystal structure of 85A, and expects another laboratory to finish up B very soon. "So once 85A is done," he said, "it will be easy to determine whether these three proteins have different immune-target epitopes, and we could design a vaccine accordingly."

The trade secret of antigen 85C - the master cell-wall builder - is its active site. This key substructure, the co-authors confirmed, controls not only constructing that strange outer envelope but M. tuberculosis' drug-resistance machinations.

It took the team the better part of three years to solve the atomic structure of antigen 85C.

Active Site Sighted At End of Tunnel

"From mutagenesis studies," Ronning recounted, "we determined that the amino acid serine 124 was pivotal for the enzyme's activity. So we looked at the area around serine 124. The active site turned out to be a hydrophobic pocket and tunnel extending 21 Engstroms into the core of the protein, which has a diameter of some 42 E. The serine is sitting right in the center of this binding pocket.

"With this tunnel that leads into the center of the protein," Ronning continued, "we've modeled in a substrate of antigen 85, and been able to fit that into the binding pocket of the crystal structure. From that we can go ahead and design more specific inhibitors.

"Our lab is doing studies right now," he concluded, "on homologous proteins in other types of bacteria that are involved in fatty-acid synthesis. Isoniazid doesn't bind to any of those. But we are using that antibiotic as a base for trying to find drugs against these other bacteria."