For most of this century, tuberculosis, or TB, has not been considered a
major disease in the United States. In the late '80s, after a century
of steady decline, the number of cases of TB in the United States began
to increase due to increases in immigrants from countries where TB is
prevalent, homeless people in crowded conditions, the elderly and AIDS
patients. Although the increase in cases has been reversed in the last
several years, more than 18,000 cases of TB were reported in the United
States in 1998. Worldwide, TB remains a serious health problem. The
World Health Organization has estimated that one-third of the world's
population is infected with Mycobacterium tuberculosis, the bacterium
that causes TB.
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."
Source :
http://www.sciencedaily.com
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