Know Your Enemy.

Scientists mine the tuberculosis genome

The threat of tuberculosis is not a new one. Traces of the disease have been found in ancient mummies, and in the mid-1800s the ailment--then known as consumption--accounted for up to a quarter of the deaths in London, New York, and Paris.

A turning point in people's relationship to consumption occurred in 1882 when the microbiology pioneer Robert Koch showed that the rod-shaped bacterium Mycobacterium tuberculosis was behind the scourge. It was the first time that a particular microbe was linked to a disease.

More than a century later, aspects of the ailment now most commonly called TB remain unexplained. Some people infected by the microbe soon show symptoms such as fever, wasting, and cough, but others don't. Unlike many bacteria, M. tuberculosis can linger in a body for decades after the initial infection without producing symptoms.

It can surge into action at any time, sometimes with deadly force. About 10 percent of people with latent infections eventually develop active disease. Researchers still don't know whether the disease persists because the bacteria lie dormant or because a slow, smoldering infection somehow escapes the body's immune response.

New genetic studies may close such knowledge gaps. In the past year or so, researchers have begun to tease out the molecular underpinnings that make the tuberculosis bacterium so infectious and enable it to evade the body's immune response. This information--including descriptions of a host of genes--may eventually lead researchers to invent new treatments and vaccines more effective than the ones currently in use (SN: 6/18/94, p. 393).

"Knowledge of one's enemy ... is an essential first step in devising a battle plan to stop the disease," says William R. Jacobs Jr. of the Howard Hughes Medical Institute at the Albert Einstein College of Medicine in New York City.

The TB bacterium isn't making it easy for scientists to go beyond those hopeful words. M. tuberculosis is highly infectious, grows slowly, is difficult to maintain in laboratory cultures, and resists several genetic techniques that have proved useful in studying other bacteria.

The incentive to circumvent these difficulties is monumental. Worldwide, tuberculosis is the leading cause of death due to infectious disease. It holds that tragic distinction although the disease is potentially preventable and, in most cases, treatable.

Standard therapy with antibiotics, however, has drawbacks. People with tuberculosis must take the drugs for 6 months to completely eradicate the bacteria. Incomplete treatment increases the risk of drug-resistant bacteria arising. Bacteria resistant to at least one antibiotic currently account for 2 percent of new TB cases in the Czech Republic, 12 percent in the United States, and 41 percent in the Dominican Republic.

Though tuberculosis is less common in developed countries than it was a century ago, it infects nearly one in three people worldwide and is especially common in those with immune disorders such as AIDS. People with active TB symptoms can spread the disease through bacteria-laden droplets they release into the air during a cough, laugh, or conversation. In most people, the immune system and the bacterial population reach a stalemate about a month after an infection begins.

One sign of this standoff is the presence of granulomas or tubercles, which are small clusters of immune cells surrounding the bacteria and infected immune cells called macrophages. While the normal function of macrophages is to engulf and kill microorganisms, M. tuberculosis relies on as-yet-unknown ways to evade destruction and persist in these cells.

"Knowledge of the molecular bases for the properties or characteristics which allow M. tuberculosis to cause disease and escape therapies is essential in developing effective new strategies" to treat and prevent the infection, Jacobs says.

Two years ago, Brigitte Gicquel and her colleagues at the Pasteur Institute in Paris became the first researchers to identify a specific gene affecting how infectious M. tuberculosis could be. Bacteria lacking this so-called erp gene, whose specific function has yet to be identified, didn't replicate as quickly as normal M. tuberculosis do in laboratory cultures or in mice.

Since then, investigators have identified several additional genes that influence the virulence of tuberculosis. Jacobs and his colleagues found three genes affecting the synthesis and export of a fatty compund found on the surface of the bacterium. Without the protein, this compound, M. tuberculosis can't infect the lungs of mice, but it can still grow in other organs.

A gene for a protein that probably helps the bacterium form its unusually thick cell wall also plays a role in enabling M tuberculosis to infect macrophages, according to a report in the February INFECTION AND IMMUNITY. At about the same time, two other papers--one published in the January INFECTION AND IMMUNITY, the other in the January JOURNAL OF BACTERIOLOGY--identified more M. tuberculosis genes that seem to help the microorganisms infect macrophages.

The list of genes continues to grow. William R. Bishai of the Johns Hopkins University School of Public Health in Baltimore has found a regulatory gene, called sigF, that helps M. tuberculosis adapt and thrive during the course of the disease. Bacteria with and without the gene multiplied at the same rate early in an infection. However, those with the gene killed the mice in 161 days, on average, while those without the gene permitted the mice to survive an average of 246 days, Bishai reports in the October INFECTION AND IMMUNITY.

Researchers are also searching for bacterial genes implicated in one of M. tuberculosis' greatest mysteries: its ability to persist in the body even while under an immune attack. "I think we're going to see a complex set of adaptive mechanisms involved in latency," Bishai says.

In the April MOLECULAR CELL, Jacobs and his colleagues identified a gene for an enzyme that modifies long fatty acids that cover the surface of M. tuberculosis and Mycobacterium bovis, a bacterium that causes TB in cows and occasionally people. By inactivating, or knocking out, the gene, the researchers showed that it's critical for continued bacterial survival and growth in infected mice. "The knockout strain grows just as well as the [normal] type during the first 3 weeks but then is unable to persist normally within the animal," Jacobs says.

During chronic, persistent infection, M. tuberculosis gets its energy from a pathway called the glyoxylate shunt. In the Aug. 17 NATURE, Jacobs, John D. McKinney of Rockefeller University in New York, and their colleagues show that without a crucial enzyme in this pathway, the TB bacterium can't persist in mouse lungs.

Two weeks after infection, mice that received M. tuberculosis that lack the gene for this enzyme, known as isocitrate lyase, had concentrations of the disease-causing bacteria in their lungs similar to those in mice infected with normal M. tuberculosis. So, the enzyme wasn't important in establishing an infection.

However, it did affect the persistence of the bacteria. After 16 weeks, the lungs of mice infected with the mutant strain showed little change. In contrast, the lungs of mice infected with normal M. tuberculosis became grossly swollen with numerous expanding tubercles.

"This was the first report of a gene that affects persistence" of M. tuberculosis in the lung, says Clifton E. Barry III of the National Institute of Allergy and Infectious Diseases (NIAID) in Bethesda, Md. He warns, however, that findings using mouse models of persistent TB may not apply to people.

"The other organs of mice with latent TB are loaded with bacteria, while in humans [with latent TB], bacterial levels are undetectable," he says.

There's a new resource helping researchers unravel the genetic clues to M. tuberculosis' capability to cause disease. Two years ago, geneticists from the Pasteur Institute and the Sanger Center in Hinxton, England, published the complete DNA sequence of the bacterium's 4,000 genes (SN: 6/13/98, p. 375). Researchers have attributed functions to roughly 40 percent of these genes on the basis of their similarities to other bacterial genes. Surprisingly large portions of the genome encode either enzymes of fatty acid metabolism, such as isocitrate lyase, or acidic, glycine-rich polypeptides of unknown function.

The genetic sequence "allows you to make a lot of hypotheses [about function] and then test them rapidly," says Jacobs. The combination of having the sequence and having the tools to manipulate bacterial genes is opening up the field, he says.

"The genome projects have been an invisible accelerator pedal for this kind of genetic research," agrees Bishai. "Months, if not years, of gene discovery can be reduced to a few [computer] mouse clicks."

In one of the hottest fields in genetics, scientists use a variety of techniques to scan an organism's genetic information to determine which genes are active in particular situations.

Using one such method, California scientists have screened about a quarter of the genome of Mycobacterium marinum, the cause of tuberculosis in fish and frogs, and found several genes that are active when the bacteria are grown in macrophages. Two of the genes encode acidic, glycine-rich polypeptides, says Lalita Ramakrishnan of the Stanford University School of Medicine. Another is similar to genes that other bacteria use to make spores and become dormant, she and her colleagues reported in the May 26 SCIENCE.

"The genome project has been the biggest boon to me," Ramakrishnan says. "It helps me decide which leads to pursue."

With another technique, scientists have identified 15 genes that are active in M. tuberculosis grown in macrophages but not in growth medium. Apparently, some interaction with the macrophages is turning these genes on, say James E. Graham and Josephine E. Clark-Curtiss of the University of Washington in St. Louis in the Sept. 30 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES.

"We're interested in genes expressed in the macrophage because we suspect these are vital in keeping the pathogen alive and destructive in the human body," says Clark-Curtiss.

The point of all this genetic research is not only to better understand the biochemical twists and turns behind M. tuberculosis' ability to infect and persist but also to use that new knowledge for developing better drugs and vaccines.

At NIAID, Barry is using genetic screens to map the TB bacterium's responses to antibiotics. Those response patterns enable him to screen for new, more effective drugs. Barry already has found several candidates that might be at least as potent as the widely used drugs isoniazid and ethambutol.

"So far, they look promising, but we're waiting for animal data," he says. McKinney, Jacobs, and their colleagues are taking another tack toward new TB treatments, using the crystal structure of isocitrate lyase as a blueprint. The researchers have developed two compounds that appear to inhibit the enzyme's function. They published their work in the August NATURE STRUCTURAL BIOLOGY.

"If we can block an enzyme that allows M. tuberculosis to persist, that might provide an avenue for attacking the bug in its latent phase," McKinney says. "A drug that targeted persistence would be quite different from conventional drugs, which target processes required for bacterial growth."

New drugs that are identified through genetic work will take years to develop, Barry cautions, adding that such medications may be critically important in avoiding drug resistance and in developing shorter, easier-to-follow treatment regimens.

Inducing the immune system to develop antibodies to proteins, like isocitrate lyase, that play a role in long-term persistence may lead to improved vaccines, says Bishai. Other targets for vaccine hunters are regulatory genes such as sigF. Disrupting the function of such genes might create weakened bacteria that could provoke an immune response without causing full-blown disease.

"Unfortunately, the completion of the genome does not guarantee delivery of safe and effective vaccines and therapies where they are needed throughout the world," Barry says. "Industry is not yet engaged actively in the search for better tuberculosis treatments. There's nowhere near the amount of activity there needs to be."

Still, the signs are hopeful, says Rick O'Brien, chief of research in the division in TB elimination at the Centers for Disease Control and Prevention in Atlanta. Researchers agree that advances in genetics and the discovery of genes important to virulence and latency point the way toward new drugs and vaccines.

In fact, O'Brien says, "this is the most exciting time for tuberculosis drug development in more than 2 decades."