VOLUME 12, NUMBER 16 • FEBRUARY 23, 2001

Using Chemistry and Genetics to Design a Global Staph Inhibitor

Staphylococcus aureus causes a wide range of illnesses, from relatively minor skin abscesses to life-threatening toxic shock syndrome and other illnesses. Antibiotics kill the bacterium, but in recent years the wily bug increasingly has become resistant to the most commonly used antibiotics. The development of antibiotic-resistant strains of Staph and other bacteria has generated widespread alarm that one day there may be many so-called superbugs resistant to all known antibiotics. But recent research from the laboratory of Associate Professor Tom Muir may provide an alternative to stopping the virulent spread of this deadly bacterium.

Tom Muir is head of the Laboratory of Synthetic Protein Chemistry.

Muir and Biomedical Fellow Gholson Lyon, working with Richard Novick at New York University School of Medicine’s Skirball Institute of Biomolecular Medicine, have combined synthetic peptide chemistry and a novel genetic strategy to develop a global inhibitor of virulence in Staphylococcus aureus.

Several years ago, Novick’s laboratory discovered that a master gene, or global regulator, regulates a signaling pathway in the bacterium that ultimately results in its release of virulent factors. It is these virulence factors that cause illness; indeed, strains of Staph that do not produce these agents inhabit our nasal passages without causing disease. Novick and co-workers later reported that a peptide, a small piece of protein, secreted by Staph activates the global regulator, and that each strain of Staph tested produced a specific peptide. Moreover, the peptide produced by one strain inhibited the global regulator of a second strain, meaning that the first strain could potentially block the virulence of the second strain.

In 1999, Novick, Muir and Patricia Mayville, a former graduate student in Muir’s lab, described the chemical structure of these peptides and synthesized molecules that can render pathogenic strains of Staph avirulent in mice, providing a novel way to disarm Staph rather than killing it outright. The ring-shaped regulatory peptide is composed of eight to nine amino acids, the building blocks of peptides, and an unusual sulfur-containing bridge called a thioester. The inhibitor compounds were designed by modifying amino acids or replacing the thioester bridge with other chemical groups, but retaining the peptides’ ring structure. In the study, the researchers also were able to dramatically weaken Staph infections in mice who had large abscesses on their backs caused by the bacteria.

The researchers knew they could inhibit virulence. What they didn’t know was if these molecules, which have the ability to switch virulence off or on, were doing it to the same class of receptor on the surface of the bacterium.

"This is important," says Muir, "because you need to know that information in order to rationally design inhibitors."

Lyon, a fourth-year student in the Tri-institutional M.D.-Ph.D. Program, joined Muir’s lab in July 1999 "looking for a project that would teach me chemistry but would also be therapeutically interesting," he says. During his medical training, he learned about the pharmacology of drug action and became interested in the process of drug discovery. Lyon worked alternately between Muir’s lab and Novick’s lab at NYU for a year and a half, learning about the microbiology and genetics of Staphylococcus aureus and the techniques of the new field of chemical biology.

Bacteria use a two-component signaling system to communicate with each other and the outside world. Staphylococcus aureus uses several proteins to positively or negatively regulate virulence: one of these, called AgrC, is the likely receptor for the peptide, and another, AgrB, is involved in biosynthesis of the peptides. Initial genetic experiments allowed the researchers to conclude that AgrC was the probable target of both activation and inhibition of the virulence response.

But, according to Muir, "You could still formally argue that it was a receptor elsewhere in the bacterial genome that we just hadn’t been able to get using the genetic tools we had. So Gholson developed a clever genetic trick that he called receptor swapping that allowed us to narrow it down to agrC."

Receptor swapping describes a technique in which the receptor from one strain of Staph, which is normally activated by one ligand and inhibited by a different peptide from another strain of Staph, is swapped to the other strain of Staph that is not normally activated by that peptide.

There are four different specificity groups of Staphylococcus aureus defined by which type of peptide they produce. A group 1 strain of Staph responds to an extracellular group 1 peptide, and group 2 strain normally is activated by a group 2 peptide. Lyon took the group 1 receptor, AgrC, and the minimal components required for signal transduction in Staph, and put them into a group 1 strain from which he had deleted all of these components, in effect replacing all the components in that strain with group 1 specificity. This strain now responded to group 1 peptide and was inhibited by group 2 peptide.

Manipulating the group 2 strain in the same way–reconstituting with the group 2 components–Lyon showed that it is activated by group 2 peptide and inhibited by group 1 peptide.

The genetic trick, says Lyon, was to swap the receptors. He took a group 2 strain, which would have all those other potential receptors, in that group 2 cell, and reconstituted the cell with group 1 circuitry. The questions were then which peptide does it respond to, group 1 or group 2, and is it inhibited by group 1 or group 2 peptide?

Lyon found that the "group 1 swap" is activated by group 1 peptide and inhibited by group 2 peptide, convincing evidence that group 2 peptide must be inhibiting virulence at the level of the receptor AgrC.

"By logical induction, it had to be the receptor AgrC, and therefore no other co-receptor or anything else in the cells conferring group specificity for inhibition and activation," says Lyon.

"The fascinating part is that a related molecule, but not identical, can bind the same receptor, but instead of switching it on, switches it off," says Muir. "That was the key bit of information we needed in designing a molecular inhibitor, because it would be much more difficult to design an inhibitor if activation and inhibition were occurring at different receptors."

Lyon and Muir reasoned that it might be possible to design a molecule that would be an inhibitor of virulence by modifying the structure of the peptides in the right way.

The peptides are composed of a ring-shaped arrangement of five amino acids, called a cyclic pentapeptide, and a tail composed of three to four amino acids. Previous analysis by Muir’s group showed that the tail component is necessary for activation of virulence. Remove the tail, and the peptide is no longer able to activate virulence. But the ring part seems to be important for both activation and inhibition.

"It appears that the ring is important in directing the peptide to the receptor, but the tail, once the peptide gets to the receptor, tells it to activate," says Muir. "We thought if we could just remove the tail, the ring should still be able to get to the receptor–but once it gets there, it doesn’t have the information to tell the receptor to activate virulence. So it will sit there, kind of like a cork in a bottle, and prevent the natural peptide from coming in and binding."

Lyon and Muir synthesized a chemical analog of the peptide, removing the tail part and replacing it with a chemical capping group. What they found was that not only does this molecule inhibit all the other groups, but it also inhibits its own group, which is a fundamental requirement for a potential therapeutic. "We’ve been able to change this from an agonist of virulence to an antagonist," says Muir.

The researchers showed that this molecule inhibits all of the four known specificity groups in Staphylococcus aureus, and thus appears to be a global inhibitor of virulence in this pathogen.

Lyon has already performed preliminary experiments on certain other species of Staph and has obtained encouraging results as well.

"This suggests to us that molecules of that general type will be inhibitors of Staph virulence–period," says Muir. "Whether or not it’s exactly this molecule, we don’t know, but this will be the template from which we can go off and find it."

Muir is head of the Selma and Lawrence Ruben Laboratory of Synthetic Protein Chemistry. This research was supported in part by the National Institute of Allergy and Infectious Diseases, part of the federal government’s National Institutes of Health (NIH), and the Burroughs-Wellcome Fund. Lyon is supported by the Medical Scientist Training Program of the NIH