By David N. Leff

Anthrax and tetanus, two potentially fatal infections, have one trait in common: Their toxic spores pass through the guts of animals - typically, horses - and then take cover in topsoil. There they lie dormant for decades until an unwary - or unvaccinated - human picks up lockjaw from the tetanus pathogen, Clostridium tetani, or ingests or inhales Bacillus anthraci, and contracts anthrax. Tetanus kills some 50,000 victims a year; anthrax, far fewer.

Far more - at least 3 million a year worldwide - succumb to infection by another pathogen, Mycobacterium tuberculosis, which also practices long-term retreat. There the resemblance ends.

"Tetanus and anthrax do undergo a kind of vegetative state," observed microbial geneticist David Russell. "They have a dormant stage, basically like a cyst, which allows them to persist for decades in hostile environments. But that's external to a host infected by TB, so it's very different. And there are few pathogens that have the level of dormancy that M. tuberculosis shows. Persistence is the hallmark of this organism."

That bacterium's life cycle is unique. When it infects a human victim, usually by being inhaled, it settles down in the lungs, awaiting its chance to strike. Like a sleeper mole in an espionage setting, the TB bug sets up housekeeping in an innocuous hide-out - the immune system's resting macrophage.

"Macrophages in the body," Russell explained, "will tend to be in the resting state. They migrate through tissues, and wander around scavenging particles of debris - essentially cleaning up around the body. When an infectious microbe comes in, and you develop a specific immune response to that infection, then the lymphocytes of the immune system release cytokines - the most important being gamma interferon - and these chemical messengers are capable of activating the macrophage."

Russell, who now chairs microbiology and immunology in the College of Veterinary Medicine at Cornell University in Ithaca, N.Y., continued: "The physiology of the macrophage will alter upon its activation, which is really a series of steps, not an all-or-nothing event. When activated to the highest level, it will acidify the vacuole in which the bacterium is residing.

Two Billion TB Carriers; 10 Percent At Risk

"During the resting stage," he went on, "the bacterium lives in this vacuole, where it will replicate quite happily. You can get 50 infectious progeny loaded inside one macrophage. But when the macrophage becomes activated, it can alter the physiology of that compartment, in which the bacterium is residing. And it can do that even with the bacterium being fully viable."

But if malnutrition, old age or AIDS weakens the body's immune defenses, M. tuberculosis quits its latent state, and pounces. One in three members of the 6 billion humans populating the planet harbors latent TB bacteria, and 5 percent to 10 percent of them have a lifetime risk of progressing to full-blown disease and death.

Russell is senior author of a multi-institutional paper in the penultimate issue of Nature, dated Aug. 17, 2000, which reports research done while he was professor of molecular microbiology at Washington University in St. Louis. His article bears the title: "Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase."

That enzyme hands the invading pathogen something like a 99-year lease on latency. Unlike bears, which hibernate in a deep sleep that drops their metabolism to near zero, latent mycobacteria find plenty to do, and this busy work requires energy and carbon nutrient.

"If bacteria were to use glucose or a carbohydrate as their energy source," Russell pointed out, "that carbon would go into the Krebs cycle, which is the process in your body by which energy and carbon are exchanged. The problem with the Krebs cycle," he went on, "is that with every turn of it you lose - you exhale - two molecules of carbon dioxide.

"The glyoxalate shunt," he explained, "is an alternative to the Krebs cycle. It's the only way we know in which the bacterium can actually retain carbon, and use it to make new carbohydrate. And glyoxalate is an organic molecule, made by that key enzyme, isocitrate lyase [ICL]."

To arraign and indict ICL as the perpetrator of the TB pathogen's persistence, the co-authors constructed two recombinant versions of M. tuberculosis - one wild type normally capable of synthesizing ICL, the other, a mutant form, ICL-deficient. Mice that they infected with the normal strain duly developed inflamed and enlarged lungs. The cohort that got the mutant bacterium steadily shrugged it off.

Animals infected with wild-type M. tuberculosis died between days 68 and 113, whereas mice inoculated with mutant, ICL-minus bacteria were surviving at day 168.

This result, Russell suggested, "looks like a reasonable and intellectually attractive drug development target. In addition, it would actually be active against a subset of the bacteria that is not targeted by any of the existing TB antibiotics."

Drugs To Come Will Aim At ICL Enzyme

In pursuit of this chemotherapeutic potential, he stated, "In practice, we've made recombinant enzymes, and are screening drug libraries made available to us by Glaxo Wellcome plc. We have several hits already," he added, "which look very encouraging. Of course there's a long way to go before development of a drug, but this does look very hopeful. What we have are basically the raw materials to start doing these drug experiments. We would imagine," he suggested, "that the compounds that would work best are those that inhibit the enzymatic action in the bacterium."

Russell is less bullish about viable immunization. "If somebody who had an HIV infection was given this as a TB vaccine," he pointed out, "it would kill him. In our last in vivo experiment with those mice," he recounted, "were gamma interferon knockouts that were infected with both the mutant and the wild-type bacteria. And eventually the phenotype of the mutant bacterium disappeared. So that a gamma interferon-minus mouse is like somebody at the late stages of HIV infection.

"If you go to central Africa," Russell went on, "and somebody comes along to the clinic presenting with TB, it's about 95 percent sure that they're HIV positive. In this region of the world, TB is the best indicator of HIV infection - like a canary in a mine.

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