Scientists supported by the National Institute of Allergy and Infectious Diseases (NIAID) have developed a technique that theoretically will allow researchers to study the function of every gene in the bacterium that causes tuberculosis (TB). The finding, reported in the Sept. 30, 1997 issue of the Proceedings of the National Academy of Sciences (PNAS), has significant implications for the development of new TB drugs and vaccines and for advancing our understanding of how TB bacteria cause disease.
"This is a major advance for the TB research community and it represents an important step in NIAID's efforts to develop research-based solutions to one of our foremost global health problems," says NIAID Director Anthony S. Fauci, M.D. "One-third of the world's population is infected with Mycobacterium tuberculosis (M.tb), the organism that causes TB. Each year, an estimated 3 million people die from TB, more than from any other infectious disease."
TB bacteria are notoriously difficult organisms to study in the laboratory. Many of the molecular techniques that scientists use routinely to analyze other microorganisms have until recently been of little use in TB research. Consequently, systematic methods for creating mutations in M.tb genes have eluded scientists. Such methods are extremely valuable in the study of disease pathogens, as they allow scientists to examine the effect of individual gene mutations on an organism's ability to grow or cause disease. These studies can reveal new drug targets or identify potential vaccine candidates.
"Analyses of M.tb have been hampered by the lack of efficient systems for transferring new genetic material into this pathogen," explains William R. Jacobs, Jr., Ph.D., senior author of the study.
"Furthermore, because M.tb is such a slow-growing bacterium, methods of creating and analyzing mutations that involve exposing cells to DNA-damaging agents, and then characterizing colonies arising from single cells, are of limited value."
Dr. Jacobs, an NIAID grantee at the Howard Hughes Medical Institute (HHMI) at Albert Einstein College of Medicine in the Bronx, N.Y., led a research team that found an efficient way to create mutations in TB genes using fragments of DNA known as transposons. Transposons insert themselves at random into bacterial DNA, inactivating any gene in which they take up residence.
Scientists have used transposon mutagenesis, as this process is called, to generate vast numbers of mutations in many other kinds of bacteria. These so-called mutation libraries allow scientists to study the function of individual bacterial genes. Until now, transposon mutagenesis has not been feasible in TB bacteria.
Dr. Jacobs, NIAID grantees Barry R. Bloom, Ph.D., also of HHMI at Albert Einstein College of Medicine, and Graham F. Hatfull, Ph.D., of the University of Pittsburgh, and their colleagues constructed special delivery vectors to carry transposons inside TB cells. Known as conditionally replicating shuttle phasmids, these vectors essentially are genetically engineered mycobacteriophages -- viruses that infect M.tb and related bacteria. For this study, the researchers developed phasmids with mutations that prevented them from replicating at a temperature of 37 degrees Celsius (C). The transposons carried by these shuttle phasmids contained a gene conferring resistance to the antibiotic kanamycin.
The researchers mixed M.tb cells with the transposon-bearing shuttle phasmids and incubated them at 37 C. Instead of replicating repeatedly and destroying the M.tb cells, at this "non-permissive" temperature the phasmids simply stuck to and entered the bacterial cells, providing an opportunity for the transposons to insert themselves into the bacterial DNA.
To determine the success of their efforts, the researchers transferred the TB-phasmid mixture to culture media containing kanamycin. Only those TB bacteria carrying the kanamycin resistance gene, by virtue of having undergone transposon mutagenesis, will grow on this media. Dr. Jacobs and his colleagues recovered thousands of kanamycin-resistant M.tb mutants in three separate experiments.
"Analyses of DNA from randomly selected kanamycin-resistant M.tb colonies revealed a random distribution of the transposon insertions," says Dr. Jacobs, "suggesting that the mutants obtained in our experiments represent libraries of independent mutants of M.tb."
In the same issue of PNAS, researchers from France's Pasteur Institute describe an alternative method for performing transposon mutagenesis in M.tb, which also results in the production of thousands of mutants. Dr. Jacobs also is a co-author of that study.
"With these techniques, researchers theoretically should be able to create mutations in virtually every gene of Mycobacterium tuberculosis," adds Ann Ginsberg, M.D., Ph.D., NIAID's TB program officer. "This should provide an unprecedented opportunity to make rapid and substantial progress in the understanding of pathogenesis and development of novel therapeutics and vaccines for TB."
In addition to Drs. Jacobs, Bloom and Hatfull, collaborators on the NIAID-funded study include Stoyan Bardarov, M.D., Ph.D., Jordan Kriakov, M.D., Ph.D., Christian Carriere, M.D., Ph.D., Shengwei Yu, and Carlos Vaamonde, M.D., of Albert Einstein College of Medicine; and Ruth A. McAdam, Ph.D., of the Central Veterinary Laboratory in Surrey, Great Britain.
NIAID, a component of the National Institutes of Health (NIH), supports research on AIDS and other sexually transmitted diseases, tuberculosis and malaria, as well as allergies and asthma. NIH is an agency of the U.S. Department of Health and Human Services.
Materials provided by NIH-National Institute Of Allergy And Infectious Diseases. Note: Content may be edited for style and length.
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