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Fighting tuberculosis one gene at a time
Scientists are searching the M. tuberculosis genome for ways to disarm
a virulent and persistent pathogen
By Sharon Guynup

Featured Article.

Over the last decade, tuberculosis has staged a powerful resurgence in humans. An estimated one in three individuals worldwide carries the pathogen Mycobacterium tuberculosis (TB) in its latent form. The bacterium is a survivor. It's been found in the tissues of ancient mummies, and new drug-resistant TB 'superbugs' are rapidly spreading among human populations, transmitted through the air during casual conversation—in a sneeze, a laugh, or a cough. Non-human strains have adapted to infect cows, birds, and other animals.

Mycobacterium tuberculosis colonies on agar.

Efforts to combat tuberculosis have intensified recently, and an essential tool for researchers is the genome sequence of M. tuberculosis. The bacterium was sequenced in 1998 at the Sanger Institute, in England, and the Pasteur Institute, in Paris. Researchers around the world are now using the information to hunt for genes that bolster the bug's metabolism and increase its virulence.

The genomic information has also facilitated a shift in strategy. For decades, the focus of research has been infection and the early stages of disease. Now, researchers want to know what happens in the later stages of infection and how to disable the pathogen.

The bug's tenacity is perhaps the biggest mystery to tuberculosis researchers. It can linger in the lungs for decades after infection, apparently awaiting a slump in the body's defenses. Once reactivated, TB explodes into full-blown disease in about 10 percent of carriers, attacking and destroying the lungs. How the bacterium survives attack by host immune cells remains unknown.

View TB infection timeline

The M. tuberculosis genome contains some 4,000 genes. Two years ago, Brigitte Gicquel, of the Pasteur Institute, discovered a gene that influences bacterial virulence, the erp gene. Strains of bacterium without the erp gene grew poorly in mice.

Since the identification of erp, researchers have found other genes linked to the bug's virulence. The pcaA gene, for example, affects the strength of the cell wall and the formation of serpentine cords of bacteria, which are characteristic of infection. Mutants lacking this gene replicated normally in mice, but they could not kill their hosts. The pcaA gene was identified by a team of researchers led by William R. Jacobs, Jr., of the Albert Einstein College of Medicine, in New York.

A tissue granuloma.

Therapies have typically targeted early infection, when bacterial cells are growing and dividing. Tuberculosis patients currently take a combination of antibiotics for at least six months—a complicated regimen of medications that costs about $16,000. This means that many people go untreated and many others drop out of treatment, opening the door to dangerous new drug-resistant bugs.

Because the body's immune response takes three weeks to kick in, cells capable of killing bacteria arrive too late to finish off the organism. By then, tuberculosis has become a persistent infection that can only be contained. Small clusters of immune cells called granulomas form, walling off the bacteria. The bugs then take up residence inside cells of the immune system called macrophages. Macrophages normally ingest invading microorganisms, but M. tuberculosis makes them a permanent home.

M. tuberculosis in mouse macrophages.

A person can harbor the bacterium for a lifetime, and antibiotics are less effective during the latent phase. For the next wave in treatment, researchers want to ferret out and kill TB where it hides—in the macrophages. "One reason to study genes involved in persistence and latency is that they will give us the ability to produce new vaccines," says William R. Bishai, of the Johns Hopkins University School of Public Health, in Baltimore.

Bishai and colleagues identified a regulatory gene called sigF that helps the TB bacterium adapt. When mice that lack the sigF gene are infected with M. tuberculosis, the bacteria thrive for eight weeks. But then they stop growing—just when the bacteria would normally begin infecting the lungs. "We think this particular regulatory gene controls late-stage survival when granulomas are forming," says Bishai. Mice exposed to normal TB live for 161 days, while mice infected by bacteria that lack sigF lived for 246 days, according to findings that were reported in Infection and Immunity.

The central puzzle in treating TB is this: In humans, months of drug treatments are required to kill the bacteria very slowly, yet the same treatments kill bacteria in petri dishes immediately. So why is TB so hard to kill inside the body? "This is a very different beast when living in the lung," says John McKinney, a geneticist at Rockefeller University, in New York. "We are looking at genes that are turned on and off in the lung."

Recombinant M. tuberculosis. Blue in center indicates where bacteria are starving.

McKinney's team of researchers discovered a gene that is key to TB's long-term survival. "There seems to be a metabolic switch that is tripped when bacteria reach the lungs," explains McKinney. The arrival of the body's immunity mechanisms about three weeks after infection triggers a change in the bacteria, allowing it to alter its diet from carbohydrates to fatty acids. The persistent bacteria uses an enzyme called isocitrate lyase (ICL) as part of a metabolic pathway that breaks down fatty acids for energy.

Mouse studies showed that bacteria without the ICL gene starve and are finished off by immune cells. "ICL is essential for the bacterium's survival once growth and division have finished," says McKinney. He and William Jacobs, of the Albert Einstein College of Medicine, knocked out the gene that codes for the ICL enzyme. Mice that received normal and knockout strains of M. tuberculosis developed comparable concentrations of bacteria in their lungs during the first two weeks after infection. But after 16 weeks, mice exposed to the mutant strain changed little; mice infected with the normal strain grew horribly swollen with granulomas. The findings were reported in Nature.

"Our hope is that a drug that targets persistence used in tandem with existing drug regimens will work better than either one of them alone, and give us a faster cure," says McKinney. But researchers estimate that a new commercial drug is at least seven years away, and an effective vaccine will take even longer to develop. Still, identifying genes that influence virulence and long-term infection are steps towards the creation of new interventions.

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Chen, P. et al. Construction and characterization of a Mycobacterium tuberculosis mutant lacking the alternate sigma factor gene, sigF. Infection and Immunity 66, 5575-5580 (October 2000).
McKinney, J.D. et al. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735-738 (2000).
Bishai, W. Lipid lunch for persistent pathogen. Nature 406, 683-685 (2000).
Sharma, S., et al. Structure of isocitrate lyase, a persistent factor of Mycobacterium tuberculosis. Nature Structural Biology 7, 663-668 (2000).
Glickman,M.S., Cox,J.S., and Jacobs,W.R. A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell 5, 717-727 (2000).

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