New Technique Identifies M. Tuberculosis Genes

United States researchers have picked up their efforts over the past decade. One, Josephine Clark-Curtiss, Ph.D., research associate professor of biology at Washington University in St. Louis, and her colleagues have recently identified 15 M. tuberculosis genes that are expressed only when the bacteria are growing in the immune system's prime gatekeeper, a disease-fighting cell called a macrophage.

Using an elaborate new technique that is widely applicable to a host of different research situations, James Graham, Ph.D., a post-doctoral researcher in Clark-Curtiss' lab, captured DNA complementary to messenger RNA (called cDNA) of genes active in human macrophages, cells that engulf and degrade pathogenic bacteria, rendering them harmless.

But in the case of M. tuberculosis, the killer bacteria have found a way, once inside the macrophage, to prevent the development of a macrophage compartment known as the phagolysosome. This key compartment produces a number of enzymes that puts the coup de grace on M.tuberculosis.

Clark-Curtiss believes the 15 genes isolated in her laboratory play important roles in the pathogen's metabolism, propagation and self-protection once in the immune system environment. Defining the specific roles of the genes could lead to drugs that target certain of the genes or even a vaccine for the disease that afflicted 6.7 million people worldwide in 1998, killing an estimated 2.4 million, according to 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 technique used allows us to study genes at different times after the mycobacteria have been introduced into macrophages. This is important because there really should be some chronology at work--genes expressed right after entering the macrophage, then some even later, and perhaps some that are involved in destroying the macrophage.

"Our next step is to construct mutant mycobacteria by inactivating these genes and infecting macrophages with the mutant strains to see which genes are critical to survival."

Clark-Curtiss published her results in the September, 1999 issue of the Proceedings of the National Academy of Sciences. Her research was sponsored by the National Institutes of Health.

The technique, called SCOTS--for selected capture of transcribed sequences--captures cDNA molecules derived from M. tuberculosis bacteria from a mixture of cDNA molecules from both the macrophages and the bacteria. Put simply, the method captures the desired cDNA molecules while discarding the unwanted ones. This is achieved by hybridization between the cDNA molecules from M. tuberculosis with chromosomal DNA from the bacteria. The hybrids are removed from the reaction mixture by magnets and the desired cDNA molecules are amplified by a widely used technique called PCR (polymerase chain reaction). In the case of the Clark-Curtiss laboratory, she and her colleagues wanted to compare M. tuberculosis cDNA grown in broth culture to that found in M. tuberculosis cDNA from infected macrophages. The technique was able to locate cDNA molecules common to both environments, but it also captured the tiny fraction of the organism's cDNA molecules that is active, or expressed, while the bacteria are in the macrophages. Magnetism, in the form of protein-coated magnetic beads, plays a key role. The beads bind the hybridized M. tuberculosis chromosomal DNA and cDNA molecules, allowing the researchers to eliminate a large portion of non-essential DNA.

The technique was developed James Graham, Ph.D., as an improvement on a cDNA subtractive hybridization technique that had been developed by Georg Plum, Ph.D., when he was a post-doctoral researcher in Clark-Curtiss' laboratory.

"We figured that there are many genes expressed in both conditions, but what we are really interested in is those genes that are expressed by M. tuberculosis when they are in the macrophages, but not expressed in broth culture," says Clark-Curtiss.

Clark-Curtiss says that two of the genes found indicate that M. tuberculosis, like many other bacteria, have a two-component regulatory system that helps them first interact with their environment, then turn on certain genes to thrive in the environment.

Other genes identified are similar to genes of other pathogenic bacteria that have been implicated as important in the virulence of those organisms. Still other genes that Graham and Clark-Curtiss identified by SCOTS have been hypothesized to be important for M. tuberculosis to grow in macrophages. But this is the first experimental evidence supporting these hypotheses.

"It's been difficult to make much progress in understanding M. tuberculosis because M. tuberculosis grows very slowly and special laboratory facilities are required because of the infectiousness of the bacteria," says Clark-Curtiss.

"Moreover, until very recently, there were no methods available to construct specific mutant strains of M. tuberculosis. The SCOTS technique allows us to compare gene expression in M. tuberculosis in response to different environments without having to first inactivate individual genes to determine the importance of each gene to growth in a particular environment.

"This technique is very useful, not just for what we're doing, but for many different situations where you want to compare gene expression in two different environments."