In recent years, health professionals around the world have noted with alarm that the drugs used to treat such diseases as tuberculosis, meningitis and pneumonia are becoming increasingly less effective as bacteria grow increasingly more resistant to antibiotics. Now, Yale biologists have developed a method that restores the effectiveness of two widely used antibiotics -- chloramphenicol and ampicillin -- by preventing the expression of the genes that make bacteria drug-resistant.
While the method has thus far been used only in the laboratory, if it is successful in animal and human studies as well, the discovery may help avert a worldwide health crisis, say the scientists.
"Although the path from our experiments to a practical therapeutic tool may be a very long and costly one, this method could restore the full usefulness of today's front-line antibiotics, thus bypassing the tremendous expense of developing new antibiotics," says Nobel laureate Sidney Altman, Sterling Professor of Biology, who announced the finding in the Aug. 5 issue of the Proceedings of the National Academy of Sciences. Senior research scientist Cecilia Guerrier-Takada and postdoctoral fellow Reza Salavati also took part in the research, which was funded by the National Institute of General Medical Sciences.
To develop the new method, the team used laboratory techniques based on Professor Altman's Nobel Prize-winning discovery that RNA is not just a passive carrier of genetic code but also actively engages in chemical reactions. The discovery triggered a new branch of genetic engineering aimed at treating lethal viruses and drug- resistant bacteria, as well as repairing genetic defects.
The Yale biologists crafted synthetic genes coding for strings of RNA and introduced them into the bacteria via small circular pieces of DNA called plasmids. The latter sometimes carry genes that cause bacteria to become drug resistant in the first place.
Once inside the bacteria, the synthetic genes produce small strings of RNA nucleotides called External Guide Sequences (EGS). The EGS's are engineered to bind to targeted "messenger RNA" (mRNA), which play a key role in controlling body chemistry. Once attached, the EGS molecules cause a RNA enzyme called RNase P to destroy the mRNA to which they are bound. The EGS molecules are then freed to repeat the process. In this study, drug sensitivity was restored in virtually all bacteria in laboratory test cultures.
The EGS technology can be used to seek out and destroy the mRNAs associated with particular diseases -- or the mRNAs associated with resistance to specific drugs. Researchers also can use EGSs to inactivate mRNA molecules in a highly selective way to gain a better understanding of how cellular chemistry functions.
"We've been working on enzymes at Yale for 25 years or more, and it was only recently that we found some potential practical value from the research," notes Professor Altman, who has been working on drug-resistant bacteria for the past six years. "You can never predict when basic investigations will yield important practical discoveries, which underscores the importance of continued support for non- applied research."
The next step in finding a practical way to restore drug sensitivity, regardless of the specific drug or infection involved, is to find the best method of getting EGSs inside bacteria, explains Professor Altman. Instead of using plasmids, which would require exposing patients to a second type of bacteria, researchers expect to identify a chemical package that can readily enter the target bacteria. Then the method must be tested in animals and humans.
It is a relatively simple matter to design the EGS sequence itself, notes Professor Altman, because the methods are "all pretty well worked out." In fact, the specific EGS that will restore sensitivity to a specific drug can be designed in a matter of hours or days, and then produced with a machine called a RNA synthesizer. The entire process of finding and testing the effectiveness of a specific EGS takes only a few weeks or months compared to years required for the development of most antibiotics.