The tuberculosis bacterium requires a specific enzyme to cause persistent infection, a consortium of researchers at Rockefeller University and three other institutions have found. The discovery suggests that targeting the enzyme could improve therapies for TB, which claims more lives each year than any other infectious disease.
The finding, published in the Aug. 17 issue of the journal Nature, resulted from a multidisciplinary effort by investigators at Rockefeller, Albert Einstein College of Medicine, Washington University in St. Louis and Texas A & M University, each of whom focused on different aspects of the problem.
The enzyme produced by the bacterium, isocitrate lyase (ICL), allows the TB microbe Mycobacterium tuberculosis to use fatty acids as a source of energy. Fatty acids are the most abundant source of stored energy in the body's cells. The new findings suggest that these reserves may be exploited by "intracellular" pathogens like M. tuberculosis, which make their living by parasitizing the cells of the host. The researchers have shown in a mouse model of TB that disabling the gene for ICL in M. tuberculosis crippled the bacterium in the disease's later, persistent phase.
"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," says lead author John McKinney, Ph.D., assistant professor and head of Rockefeller's Laboratory of Infection Biology. "A drug that targeted persistence would be quite different from conventional drugs, which target processes required for bacterial growth. ICL is not present in humans and other vertebrates, so blocking its activity should not cause harmful side effects in the host."
When M. tuberculosis infects the body, the immune system rallies to fight the invader but is unable to eradicate the microbe completely. A stalemate is achieved in which the bacteria continue to live in infected tissue, although they are not multiplying. The infection can persist in a latent or chronic state for the lifetime of the host and can flare up if the immune system is later weakened.
The bug's persistence in the body has been the main barrier to clearing the TB pathogen with drugs. Current treatments require three to five drugs for at least six months, a regimen many patients are unable or unwilling to follow. The drugs can have unpleasant side effects and in some cases cannot be taken because they interfere with other medications. Consequently, most patients fail to complete therapy unless their compliance is closely monitored.
"A drug that inhibits ICL activity or synthesis might allow us to shorten the duration of chemotherapy from six months or more to just a few weeks," McKinney says. "That would have an enormous impact on patient compliance and cure rates."
The multidisciplinary project grew out of research that McKinney initiated as a postdoctoral fellow in the laboratory of William R. Jacobs, Ph.D., a bacterial geneticist at the Albert Einstein College of Medicine and investigator with the Howard Hughes Medical Institute. Jacobs and McKinney, a 1994 Rockefeller University graduate, attacked the problem using bacterial genetics and animal infection models. Co-author David Russell, Ph.D., formerly of Washington University and now at Cornell University, focused on enzymology and cell biology. James Sacchettini, Ph.D., of Texas A & M University, solved ICL's physical structure using the imaging technique known as X-ray crystallography.
In the Nature paper, the researchers demonstrated ICL's link to persistence of the TB bug by infecting mice with two strains of M. tuberculosis-one containing the ICL gene and a mutant strain that lacked the gene. During the early "acute" phase of infection, growth of the two strains was identical. Once infection entered the "chronic" phase, however, the mutant strain was largely eliminated by the immune system while the wild-type strain maintained peak loads in the animals. Not surprisingly, the mutant bacteria were less virulent. "We're keeping mice that were infected with the mutant bacteria more than a year ago-they're still alive and vigorous," notes McKinney.
The research suggested that M. tuberculosis reprograms its carbon metabolism in response to the activation of certain immune cells-called macrophages-in the host. This idea was confirmed in subsequent experiments. Russell and colleagues showed that the mutant bacteria were unable to survive in immune-activated macrophages, yet grew normally in macrophages that were not activated. In parallel, McKinney and colleagues found that when the mutant bacteria were injected into immune-deficient mice, their virulence was restored.
"This is an unusual finding-that the immune response of the host dictates the basic carbon metabolism of the pathogen," McKinney says. "I don't know of any precedent for this in the literature."
A separate paper, published by the same consortium of investigators in the August issue of Nature Structural Biology, reveals the three-dimensional structure of ICL. The work was led by structural biologist James Sacchettini, whose group also determined the structure of ICL in complex with specific inhibitory molecules that blocked the ability of the bacteria to use fatty acids as an energy source. Knowledge of the enzyme's shape is guiding current efforts by collaborators at the Glaxo Wellcome Medicines Research Centre in Stevenage, UK to identify drugs that will inhibit ICL's function. These efforts, led by Ken Duncan, Ph.D., have already paid off handsomely, with several novel ICL inhibitors already identified and under study.
McKinney says the collaboration among the scientists at the various institutions was the only way the TB research could progress so quickly.
"None of us working alone could have gotten us where we are. Tuberculosis R&D is slow, difficult, and expensive, and it's not a high priority in the U.S. because it mostly affects people in developing countries," he says. "Drug companies are understandably reluctant to invest a lot of money up front in basic research on TB. But by attacking the problem simultaneously from many angles, our consortium has made ICL an attractive target, and in just a few years we've moved from an idea to having leads that could form the basis of a full drug-discovery program. Considering that there have been no new drugs developed specifically for TB since the 1960s, that's not too bad."
TB looms as a major public health issue that is intensifying. About 16 million people worldwide have active TB, and an estimated eight million new cases occur each year. Nearly two billion individuals-a third of the global population-are believed to harbor the infection in its latent form. In the United States and elsewhere, the spread of the human immunodeficiency virus (HIV) and the compromised immune systems that result have greatly increased the number of people susceptible to the TB germ. A drug that targeted persistent bacteria could be used as an adjunct to conventional treatments to clear TB infections much more quickly. That could translate into millions of lives saved every year.
Support for the research reported in the Nature and Nature Structural Biology papers was provided by Glaxo Wellcome and a Program Project grant from the National Institutes of Health. In addition, the Helen Hay Whitney Foundation and the Howard Hughes Medical Institute supported the research reported in Nature, and the U.S. Department of Energy and the Robert A. Welch Foundation supported the research reported in Nature Structural Biology.
John D. Rockefeller founded Rockefeller University in 1901 as The Rockefeller Institute for Medical Research. Rockefeller scientists have made significant achievements, including the discovery that DNA is the carrier of genetic information. In 1999 cell biologist Günter Blobel won the Nobel Prize for medicine for his discovery that proteins are customized with intrinsic "ZIP codes" for delivery to exact destinations within the cell. The University has ties to 20 Nobel laureates. At present, 32 faculty are elected members of the U.S. National Academy of Sciences, including the president, Arnold J. Levine, Ph. D. As it anticipates its centennial anniversary in 2001, Rockefeller-the nation's first biomedical research center-continues to lead the field in both scientific inquiry and the development of tomorrow's scientists.
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Nature