Sublethal Force: New Antibiotic Aims to Tame Bacterial Toxins

Disarming Cholera Takes Punch out of Pathogen

Using an innovative screening approach, researchers in the lab of John Mekalanos have identified an entirely new class of antibiotics active against the cholera bacterium. While traditional antibiotics kill bacteria outright by interfering with processes essential for their survival, the new agents block production of bacterial proteins that promote infection and cause cholera symptoms. Tests in animals proved that the new compounds could prevent bacterial colonization.

Photo by Steve Gilbert

The screen team, consisting of (from left) Elizabeth Shakhnovich, project leader Deborah Hung, lab head John Mekalanos, and Emily Pierson, targeted the pathway that controls cholera-toxin production to discover a new kind of anticholera antibiotic.

The work opens up a new world of potential for antibacterial drugs that aim to block the unique disease-causing talents of cholera bacteria, which include the production of cholera toxin. “What we’ve done is made a custom, organism-specific antibiotic against Vibrio cholerae,” said Mekalanos, the Adele Lehman professor of microbiology and molecular genetics and head of that department. Since most bacteria that cause human disease elaborate virulence factors such as toxins, Mekalanos said, “There is no reason our approach can’t be replicated for a number of other important pathogens.” The research appeared Oct. 13 in the online edition of Science.

Antibiotic Action
For a while at least, science seemed to be winning the battle against disease-causing bacteria. With the development of modern antibiotics, formerly fearsome foes were vanquished with a pill from the pharmacist. But the bugs fought back, and the spread of antibiotic resistance among many common pathogens is causing a major public health problem today.

Most of the antibiotics now available shoot to kill, taking aim at bacterial growth and survival. New antibiotics are, for the most part, redesigned versions of the old ones, tweaked to avoid resistance and restore potency. But rather than escalate the arms race with more of the same deadly force, cholera maven Mekalanos and Deborah Hung, an instructor in medicine and a physician in the BWH Division of Infectious Disease, chose a different plan of attack.

They knew that Vibrio cholerae, the bacterium that causes endemic cholera in many parts of the world, makes people sick by invading their intestine, where it produces the cholera toxin protein. This toxin stimulates a massive outflow of fluid and salts from the intestinal lining, and the resulting diarrhea can kill if left untreated. Over the last 25 years, research in the Mekalanos lab has resulted in a detailed understanding of the role of cholera toxin in disease and especially how the production of toxin is regulated at the genetic level.

Enter Hung, trained as a chemist and eager to apply the tools of chemical biology to the problem of developing a new anticholera drug. Hung devised a high-throughput screen for inhibitors of cholera toxin–gene expression and ran through a library of 50,000 small molecules at the HMS Institute for Chemistry and Cell Biology. Then Hung, working with HMS graduate student Elizabeth Shakhnovich and research assistant Emily Pierson, selected 15 promising compounds and confirmed that they had no effect on bacterial growth in a test tube. Further in vitro studies on one candidate, which they dubbed virstatin, revealed that it not only inhibited cholera toxin–gene expression, but also turned off the production of other proteins that allow cholera bacteria to stick to intestinal cells. Virstatin worked by inhibiting a trans-cription factor responsible for the coordinated regulation of several virulence-associated genes.

In Vivo Success
To check if blocking virulence-factor expression in vitro translated into inhibition of disease in vivo, the researchers turned to a mouse model of cholera infectivity. Sure enough, when they treated mice with virstatin, followed by a dose of Vibrio cholerae, the bacteria failed to gain a foothold in the intestine. When virstatin was given a day after infection, the bacteria could still be dramatically reduced, suggesting that virulence inhibitors may be useful for either prevention or symptomatic treatment.

While the idea of blocking virulence is not new, this is the first time researchers have successfully used a screening approach to identify and attack new targets in virulence pathways. “We didn’t have a particular protein target and we had no chemical structural information to begin with. We just said, here’s the endpoint, can we find something in a screen that will inhibit the end result of cholera-toxin production,” Hung explained.

Their novel approach almost guaranteed that they would identify the bacterium’s Achilles heel, since the best inhibitors are the ones that hit the weakest link in the pathway. Mekalanos said he hopes that their results might inspire others to undertake similar efforts in other organisms. “For nearly every bacterial pathogen anyone has looked at, there is a regulatory protein controlling expression of virulence factors, so this is a logical target to propose,” he said. “Simply put, for cholera, we picked an arbitrary target, and it worked, so now that argues that anyone else who picks an arbitrary target probably can have a pretty good chance of success, too.”

Targeting virulence factors will not eliminate the problem of resistance, but new antibiotics could help by sparing the use of older, resistance-prone drugs. The researchers showed that a simple mutation of virstatin’s target protein could render the bugs immune to the compound’s action. But even so, because virstatin acts on a cholera-specific pathway, the resistance cannot spread to other organisms, a big problem for many current drugs. And because the mechanism by which virstatin works is different from that of any other antibiotic, it presents the possibility of potent combination therapies.

The present work results from more than 20 years of basic science on cholera pathogenicity, much of it carried out in the Mekalanos lab, said Matthew Waldor, a former postdoc and current faculty member at Tufts University. “Mekalanos and Hung have now taken all that knowledge about the bacteria and put it together to devise a screen to identify compounds that inhibit production of cholera toxin,” Waldor said. “This idea that turning off virulence genes rather than killing pathogens could lead to a new type of antibiotic has been kicking around for a while, but in this case, the proof of the pudding is in the tasting—they made it work. They’ve created a new paradigm for developing antibiotics.”

—Pat McCaffrey

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