Malaria Parasites Can Hide From Our Drugs. Now We Know Why.

When Jennifer Small-Saunders started her malaria research, “the little rings” she kept seeing in the blood smears under her microscope after she treated the parasites with an antimalarial were an enigma.

Artemisinin, the core component of malaria combination therapy, usually kills parasites within hours. “All you’ll see are these really small dots of dead parasites in the infected host cells,” says Small-Saunders, now assistant professor of medicine in the Division of Infectious Diseases at Columbia University Vagelos College of Physicians and Surgeons.

The “little rings” Small-Saunders saw were parasites that survived the artemisinin exposure and reached the “ring stage,” when they begin growing rapidly.

The surprise wasn’t that the parasites survived: these were parasites armed with a mutation that confers resistance to artemisinin. What was perplexing to Small-Saunders was the variability in response she saw among genetically identical parasites.

"The resistant parasites had the same genes and should have responded to the drug in the same way. But every time we ran the assay some would survive, and some wouldn’t,” Small-Saunders says. “Something was going on beyond the genome that was letting some persist.”

That something, she has found, has opened a new window into malaria biology, revealing how the Plasmodium falciparum parasite can use an ancient epitranscriptomic mechanism to hide from today's antimalarial drugs.

“I think this is one of the most important papers in malaria research in recent years,” says Small-Saunders’ mentor, David Fidock, who’s been working in the field since the 1990s. “It answers two of the biggest mysteries surrounding artemisinin: How is the parasite able to resist these drugs and how does this relate to artemisinin-induced dormancy?”

Artemisinin acts like a “stress bomb”

Small-Saunders joined the Fidock lab as a postdoc after finishing her infectious diseases fellowship at Columbia. Malaria parasites were becoming increasingly resistant to the antimalarial artemisinin and her goal was to learn how some parasites can evade the drug and use that information to help lead the way to new therapeutic strategies.

Though a malaria vaccine is now available for children in some African countries, new antimalarial drugs are still needed. “Vaccines won't replace the need for drugs,” says Fidock, who was part of the WHO committee that approved the vaccine. “The vaccine on its own has modest efficacy; it’s only when you combine it with drugs that we see a substantial benefit.”

Artemisinin has been a successful antimalarial because “it acts like a stress bomb,” Small-Saunders says. “It doesn’t hit a specific target, it oxidizes all sorts of molecules—proteins, lipids, DNA—and basically causes so much stress that the parasite can’t cope, leading it to ­­­stop developing and die.”

But some parasites can now survive the onslaught. The fact that all the parasites in her assays were genetically identical meant that what allowed some to survive must be epigenetic, something not written in the parasite’s genetic code.

And that something had to act fast, because artemisinin starts wreaking havoc inside the parasite within minutes.

“Just in time” tRNA reprogramming

The requirement for a rapid defense suggested to Small-Saunders that the parasite might be using a common stress coping response in organisms: tRNA reprogramming.

When the cell adds or removes modifications to transfer RNAs, these mediators of protein synthesis can be reprogrammed to prefer some codons over others, which pushes the composition of the cell’s protein repertoire (or “proteome”) in a certain direction.

“tRNA modifications tap into a code that lets the cell manipulate the speed at which certain proteins are translated in favor of others,” Small-Saunders says. “Those with the preferred codons get translated faster, and within minutes the cell can have a different array of proteins.”

“It’s a very clever system, often used in organisms from yeast to cancer cells to cope with stress,” Fidock says. “And it is very fast as it bypasses the need to mutate the genome; it’s often called a ‘just in time’ stress response.”

The first clue that malaria parasites might use tRNA modifications to survive exposure to artemisinin came when Small-Saunders applied new mass spectrometry techniques to analyze the parasite’s tRNA repertoire, in collaboration with scientists at MIT and Nanyang Technological University. These experiments revealed that after drug exposure, tRNA modifications were globally reduced in surviving parasites, while little changed in drug-sensitive parasites.

What then convinced the researchers was a codon analysis of every increased and decreased protein in the surviving parasites. “If the tRNA reprogramming changed tRNA codon preferences, we should see a change in codon usage in each protein compared to the genome’s baseline percentage,” Small-Saunders says. “Our studies identified a pretty significant codon bias in parasites that had survived the drug exposure.”

The researchers were even able to make a drug-sensitive parasite resistant to artemisinin simply by preventing one class of tRNA modifications.

“Essentially what happens is that the tRNA reprogramming quickly changes the proteins active inside the parasite, putting it into a quiescent state that allows parasites to survive drug-induced stress,” says Small-Saunders. “Artemisinin only works for a few hours, so this defense mechanism is a perfect way to wait out the storm.”

New drugs targeting tRNA modifications for malaria?

Small-Saunders, who has recently started her own laboratory at Columbia's Center for Malaria Therapeutics and Antimicrobial Resistance, has begun screening compounds and has already identified novel tRNA modifier inhibitors that can kill parasites. “I think it's very feasible that these pathways can be targeted, and we’re already seeing that in other fields, especially cancer,” Small-Saunders says.

The study has revealed a new layer of complexity in the malaria parasite that could be mined for additional drug targets.

“We're always looking for new parasite biology that we can, hopefully one day, target,” Small-Saunders says. “And now we’ve opened this whole unexplored area of epigenetics and epitranscriptomics that we’ve never thought about before.

“I think this is where my physician-scientist training comes in handy,” she adds. “Epigenetics and transcriptomics are well known mechanisms in the cancer biology field. But in the microbial parasite world, it's an entirely new area of therapeutically relevant research. All these elaborate just-in-time stress responses that are going on in other cells, they're probably happening in our microbial and parasitic infections, too, and we can now start exploring them.

"Our hope is to leverage this information to reduce the burden of malaria, where still over 600,000 individuals, mostly young children in Africa, die every year.”