One day in the future, infection may be fought by simply switching bacterial invaders off. At least, that's the promise of new technology out of a group at Yale University that's studying riboswitches--short sections of untranslated RNA that monitor small compounds in the cell-like nucleotides, amino acids and sugars--in order to control gene expression. This nascent technology, which is currently being tested on simple bacteria in the lab, may soon constitute a novel class of antibiotics, those wonderful "magic bullets" from the 20th century that suddenly are encountering resistance from evolving bacteria.
The majority of antibiotics thwart the bacterial cell by targeting either ribosomes to stop protein synthesis or the proteins involved in DNA replication. Some antibiotics work by interfering with the biosynthesis of cell walls, or with folate--a form of vitamin B integral to the maintenance of new cells. "There's no method addressing RNA-mediated gene regulation," notes Kenneth Blount, a postdoc researcher in cell biologist Ronald Breaker's lab and the first author on the riboswitch study, published in this week's issue of Nature Chemical Biology. Breaker's group sought to exploit riboswitches, which they first characterized in 2002. In the current study, they created variations in the amino acid lysine to target its class of riboswitch. "The drug compounds, if they're a good enough mimic of that metabolite, bind to the riboswitch and trick the cell into thinking that it's swimming in the metabolite, that it's rich in the metabolite, when in fact it's starving for it," Breaker explains. If the riboswitch believes there is an excess of lysine in the cell, it will shut off its production. Without lysine available, the bacteria will be unable to translate its RNA into proteins, which will halt its growth.
To accomplish this chemical deception, the Yale group started with a lysine molecule and made slight chemical modifications. These changes ran the gamut from replacing a carbon in its backbone with a sulfur or oxygen atom to attaching bulky groups on its end. The group then tested each version in a common soil bacterium, Bacillus subtilus, to see whether the lysine riboswitch would bind to them while the rest of the cell would ignore it, knowing that it wasn't actually the amino acid. The three versions that bound best involved the substitution at the position of the fourth carbon in the lysine chain. "It's sort of like a lock and key mechanism where there are a few positions where the riboswitch does not have a tumbler," Blount explains. "But there are other positions where if you change the key, it doesn't fit." Oddly enough, these configurations proved the most effective in quelling bacterial growth.

To test whether the growth inhibition was a result of efficient binding, the group grew bacteria in a drug-rich medium, so that the strain could build up a resistance to the imposter lysine molecules. When they introduced all of their compounds into these mutant bacteria, they found that some cells indeed survived. After sequencing the live cells, they determined that these cells had mutations to their lysine riboswitches. In fact, according to Blount, they "mutated in such a way that the riboswitch can no longer bind as well to the drugs and they also mutated in such a way that the riboswitch can no longer shut off genes that are controlled by that switch." Breaker adds: "This really strongly implicates the riboswitch as the target for those drugs."
Gerard Wright, a biochemist at McMaster University in Hamilton, Ontario, says this work by Breaker's group is essential to fighting the growing antibacterial resistance: "There's a whole series of small, noncoded RNAs in the microbial genome that we really don't understand what they do. And a number of these are likely going to be riboswitches."

Breaker, who has a feature article on riboswitches in the January 2007 issue of Scientific American, cautions that he conducted his study under very specific conditions and that the results are not yet applicable to treating infection in animals: "We can wonderfully cure plastic test tubes of bacterial infection. But it's much more challenging when you get into a patient." In the human body, for instance, bacterial cells will be surrounded by a rich medium that will likely include multiple sources for lysine, so it will not have to provide the amino acid for itself. Though, he notes, many classes of riboswitches can serve as targets, including those for derivatives of vitamins such as riboflavin and B12 as well as other amino acids. Still, looking toward the future, Wright notes, "The field needs new approaches, and this is one of them." --Nikhil Swaminathan