MRSA, being engulfed by a white blood cell. Credit: NIH
MRSA, being engulfed by a white blood cell. Credit: NIH

Killing Sleeper Cells and Superbugs with Assassin Janitors

For years, scientists and science writers have been sounding loud alarms about drug-resistant bacteria that can shrug off our most potent antibiotics. They are hard to kill, and so kill us with greater ease. These superbugs are undeniably worrying but they’re only part of the problem—it is entirely possible for bacteria to defy antibiotics without actually resisting them.

All of our antibiotics are designed to kill fast-growing microbes. Bacteria can weather these assaults by entering a dormant state, and waiting until the drugs have worn off. These sleeper cells are called persisters. They’re the reason why bacteria sometimes cause long-lasting infections that repeatedly bounce back despite our best attempts to treat them. Each wave of drugs wipes out most of the microbes, but a small group of persisters can survive to re-start a new wave of infection.

Persisters are not drug-resistant; if you whacked them with antibiotics while they were growing, they’d die. But give the same drugs to a patient, and not much happens. The bacteria survive because of their dormant nature, because of how they behave rather than what they are.

We’ve known about persisters since the 1940s, but they are hard to study and to kill. Many scientists are tackling the issue of drug resistance but persistence has, ironically enough, lain largely dormant.

But Kim Lewis from Northeastern University has now found an exciting way of killing persisters, with an antibiotic called ADEP4 that forces these cells to eat themselves in their sleep. He hasn’t tested it in humans yet, but it can completely clear severe and long-lasting infections in mice. It even kills persisters that are also resistant to traditional antibiotics, such as MRSA. It’s a very important milestone,” says Lewis.

Lewis has been plugging away at the persister problem for years. It has been a difficult journey. “We were frustrated because we couldn’t kill them with anything,” he says. This team initially tried to discover how bacteria enter the persister state, to work out ways of preventing this transformation. But they quickly discovered that there are many routes to persistence. The common gut microbe E.coli has at least ten of them. Disrupt one, and they can head down many others. “That told us that conventional drug discovery approaches simply won’t work,” says Lewis.

He changed tack, and instead searched for chemicals that could kill inactive cells. That led him to ADEP4. Discovered in 2005 by scientists from Bayer Healthcare in Germany, ADEP4 killed a variety of different bacteria and cured lethal infections in mice and rats.

Here’s how it works. Proteins need to fold into very precise shapes to do their jobs, and misfolded proteins are wastes of space. Bacteria dispose of these useless molecules with ClpP—a janitorial protein that digests other proteins. It works with a partner, which recognises misfolded proteins, unfolds them, and threads them through a hole in the middle of ClpP so they can be broken down. But ADEP4 opens ClpP up so it no longer needs its partner. The janitor now becomes an assassin, running amok and chopping up any protein it comes across, misfolded or not.

The Bayer scientists showed that ADEP4 can force fast-growing cells to self-destruct, but Lewis suspected that it would do the same to persisters. Afterall, ClpP’s partner requires energy to do its job, but ClpP itself doesn’t. Once ADEP4 opens it up, it should go about its fatal business even in a dormant cell.

Lewis’ team found that ADEP4 did effectively kills persister populations of Staphylococcus aureus, but the bacteria bounce back. ClpP isn’t essential, so the bacteria just inactivated it to evolve their way around ADEP4. This, says Lewis, is why Bayer stopped working on the drug.

His solution was to pair ADEP4 with another antibiotic called rifampycin. ADEP4 would kill off the majority of the persisters, and if any of the rest started growing again, rifampycin would finish them off. He predicted  that the double-whammy would leave very few survivors, maybe just a thousand cells or so.

“That’s not what we saw,” he says. “What we saw was complete sterilisation.”

It turned out that while bacteria can easily lose ClpP, the mutant strains are very wimpy. “They become susceptible to killing by almost anything,” says Lewis.

The team tested the combo of ADEP4 and rifampycin against several strains of S.aureus persisters, including three that are also resistant to antibiotics. One is among the most common strains of MRSA in the USA, another causes virulent bone infections, and a third came from a patient who had previously died of an antibiotic-resistant infection. ADEP4 and rifampycin eradicated all three of them.

They also managed to finish off biofilms—slimy microbial cities that bacteria create and live within. Biofilms cause deep-seated infections when they form in organs like the heart or airways, and they can stubbornly contaminate medical equipment like catheters. They are notoriously hard to destroy, but Lewis’s drug combo left no traces of biofilms after just 24 hours.

As a final test, the team tested the drugs in mice. They crippled the rodents’ immune systems and exposed them to large doses of S.aureus, creating severe, deep-seated infections of the type that plague immuno-compromised human patients. Antibiotics like vancomycin and rifampycin could kill some of the bacteria, but never managed to clear them. But rifampycin and ADEP4 sterilised the infected tissues in just a day. When exposed to these drugs, the persisters could not persist.

“I’m particularly impressed with those mouse experiments,” says Bruce Levin from Emory University, who studies persisters. “It’s a major find, and I hope that somebody’s developing ADEP4 further.”

Lewis is on the case. He is working with a group of chemists to produce even more effective versions of the compound, and with a biotech company called Arietis to test the drug and usher it into clinical trials.

ADEP4 isn’t a magic bullet. For a start, it only works the bacteria known as gram-positives, which includes problematic bugs like Staphylococcus, Streptococcus and Clostridium. It is too big to pass through the extra outer layers of the gram-negative bacteria like E.coli, Salmonella or Pseudomonas. But Lewis thinks that he should be able to find other smaller compounds that target ClpP or similar janitorial proteins. Now that he has found one way of killing persisters, it should be easier to find more.

Mark Brynildsen from Princeton University says that the discovery is a “significant contribution”. In 2011, he identified another possible way of killing persisters: by persuading the sleeping cells to take up chemicals called aminoglycosides that eventually kill them. This method also killed biofilms, and helped to treat chronic infections in mice. “These studies both suggest that stimulation of activity in persisters is the way to go, to discover treatments for chronic infections,” he says.

Reference: Conlon, Nakayasu, Fleck, LaFleur, Isabella, Coleman, Leonard, Smith, Adkins & Lewis. 2013. Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature

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