In the 1700s, French astronomer Jean-Jacques d’Ortous de Mairan noticed that the leaves of the mimosa plant opened towards the sun and closed at dusk. His discovery was in keeping with thousands of years of observations. But de Mairan also found that the plant followed the same rhythm even in the constant darkness of a cupboard, suggesting that some innate metronome kept the plant in sync with the rotation of the earth. Centuries later, scientists now know this to be a circadian rhythm, a 24-hour cycle that readjusts in response to environmental cues such as sunlight.
When Filipa Rijo-Ferreira, then a graduate student at the University of Porto in Portugal, first read about circadian rhythms, it seemed they set the clock for just about everything. Studies show that wounds heal faster during the day, and patients over 65 produce more antibodies if they get a flu shot in the morning. Disruption of circadian rhythms has been linked to sleep disorders, diabetes, Alzheimer’s, and cancer.
Plants, animals, fungi, and cyanobacteria have all independently evolved circadian clock genes to anticipate changes in their environments by controlling when cells express other genes. Until recently, scientists hadn’t looked for those genes in parasites, in part because they live inside hosts like humans and don’t experience daylight. And while there were hints they might run on a similar clock — malaria patients, for example, develop fevers every 24, 48, or 72 hours — most researchers assumed that parasites respond to changes in their hosts.
“I started thinking that this might not be the full story, that perhaps the parasites themselves could have evolved a way to really anticipate those rhythmic changes of their world,” said Rijo-Ferreira, now an assistant professor of infectious diseases and vaccinology at the University of California Berkeley.
Rijo-Ferreira, who was recently named a STAT Wunderkind, is part of a small group of researchers behind an emerging body of evidence that parasites have circadian clocks, too. She wants a clear-eyed view of these rhythms: how they shape the back-and-forth between parasites and their hosts, how the ticking of one clock changes the time of another — and how scientists might one day disrupt them to improve treatments of disease.
Rijo-Ferreira grew up in Almada, Portugal, where the Tagus River empties into the Atlantic. The city’s parks and long coastline are broad-strokes beautiful, but Rijo-Ferreira dreamed small. In parking lots, she squatted beneath the branches of trees, gathering leaves, flowers, and ants to bring up to her family’s apartment. Back home, she would study her collection under a toy microscope her father, a businessman at a pharmaceutical company, had brought home from work. She didn’t know what she was looking for, but she loved watching the world move.
In high school and university, Rijo-Ferreira’s curiosity about her surroundings crystallized into an interest in microbiology. When she was accepted into a Ph.D. program at the University of Porto, she approached Luísa Figueiredo, a parasitologist studying sleeping sickness at Lisbon’s Institute of Molecular Medicine. Transmitted by the tsetse fly, sleeping sickness is endemic in 36 sub-Saharan African countries and is almost always fatal if untreated. Rijo-Ferreira had spent the last year after finishing her undergraduate degree working as a tech in Figueiredo’s lab, trying to determine how the Trypanosoma brucei parasites evade the immune system before crossing from the bloodstream into the brain.
But she was intrigued by the disease’s neurological symptoms, the way patients sleep a normal amount but at unusual times of day. She wanted to know how the parasite disrupted their natural rhythms.
Figueiredo herself had idly posed the same question at the end of a department seminar, more as a conversation starter than a potential avenue of research. Pursuing the idea would mean starting from square one: No one had studied how parasites disrupt sleep, so there were no models to borrow or experiments to replicate. “[Rijo-Ferreira] would go into PubMed, and there was almost nothing to read,” Figueiredo said.
Instead, Rijo-Ferreira read all she could on sleep and stumbled upon circadian rhythms for the first time. She became convinced sleeping sickness could have a circadian component and enlisted the help of Joseph Takahashi, a neuroscientist and geneticist at the University of Texas Southwestern Medical Center. Under the combined guidance of Figueiredo and Takahashi, who would later become her postdoc mentor, she began studying the gene expression of T. brucei. Rijo-Ferreira grew the parasites in an incubator with a cycling temperature to mimic conditions in the host: Human bodies fluctuate in temperature at different times of day. Once she had “trained” the parasites to a 24-hour cycle, she kept some on the cycle and others at a constant temperature.
For 24 hours, Rijo-Ferreira ignored her own circadian rhythms and collected parasite samples every four hours, curling up on the navy blue futon outside the lab between time points. She’d had help with early-morning visits when training the parasites — Figueiredo and even the cleaning staff had helped transfer flasks between incubators — but now she was on her own.
“One thing that I really love when doing experiments is really the planning,” she said. “And so everything was so, now, well planned, that it was just executing it.”
What she found was striking. Nearly 1,500 genes had a 24-hour cycle, increasing or decreasing expression levels at certain times of day. More than 1,000 of them oscillated even without temperature cues, like a plant opening its leaves towards a sun it can’t sense.
The parasite, it seemed, was working on its own time.
As a postdoc, Rijo-Ferreira’s experiments became more elaborate, her models expanding to include the animals playing host to the parasites. She found that mice infected with T. brucei showed symptoms of sleeping sickness, running on their wheels during the day rather than at night, as they usually do, and for shorter periods.
Next, she turned to the Plasmodium parasites, which cause malaria — a disease that sickens hundreds of millions of people every year, claiming 627,000 lives, almost entirely in Africa, in 2020 alone.
To study the gene expression of malaria parasites in mice, Rijo-Ferreira needed light rather than temperature to synchronize the animals’ clocks. And to be certain that she was observing the cycling of clock genes rather than a response to light, she’d need to collect blood samples and infect mice under constant darkness. She strapped on night-vision goggles and got to work.
The goggles were cumbersome and, like a microscope, only focused on a specific range. “Whenever I would try to focus for what I thought would be the right distance, but then I would have to literally put my hands at that distance so I could see sharply for collecting the blood or for initiating infections,” Rijo-Ferreira said. “You’re with needles and parasites and it’s actually not the easiest. [The goggles] are heavy. And they start also hurting your nose.”
With time, maneuvering in the dark became easier. When she emerged to analyze her samples, Rijo-Ferreira found that Plasmodium had more than 4,000 cycling genes. These rhythms persisted for every possible clock-setting influence she tested in mice — whether the animals lived in constant darkness or fed constantly, rather than in spurts. The cycles even endured inside mice with disabled clock genes.
“Almost the entire genome cycles in the parasite,” Takahashi said. “It’s amazing.”
In the 25 years since his group discovered the first mammalian clock gene, Takahashi has watched his field grow. Scientists in other disciplines continue to discover circadian rhythms in new organisms or aspects of human health. He wasn’t surprised to find that Plasmodium had them, too.
But the infectious disease community, he said, was shocked. The results suggested a complex synchrony between the rhythms of parasites and their hosts. A clock that has evolved to match its environment could help parasites infect a host when more nutrients are available, or during periods when the host’s immune system is less active. It could also help the parasites thrive during times when vectors such as mosquitos are more likely to bite a host. Rijo-Ferreira’s discovery, Takahashi said, gave the community a new way to think about how to treat disease.
In her new lab at Berkeley, Rijo-Ferreira is eager to discover the clock genes of parasites that drive the rhythms she has observed. With that knowledge, she could then disrupt those genes and see how parasites behave in response. “Once we are able to then disrupt the molecular clock of parasites, we are also potentially tilting the scale to benefit the host,” she said.
“It’s as if she always has the eyes open and she’s in the world, interacting with the world.”
Luísa Figueiredo, parasitologist
Already, Rijo-Ferreira has found that T. brucei is 2.5 times more sensitive to suramin, a drug used to treat sleeping sickness, at certain times of day. Few clinical trials account for timing of treatment, but there are signs that doing so could have broad benefits. Most drugs target genes with cyclic expression patterns. A recent analysis of about 100 trials that examined drug toxicity or treatment efficiency at different times found that 75% reported different responses based on time of day. Including time as a variable can make clinical trials impractically large, but Rijo-Ferreira said that even recording when patients receive a drug could help researchers compare the timing of other trials afterwards.
For now, Rijo-Ferreira is focused on pinning down clock genes in Plasmodium and T. brucei, but hasn’t ruled out turning to a new parasite when she has the time. She wants to know how many other parasites have their own rhythms that might impact infection, and whether circadian clocks underlie some of the patterns parasitologists have observed for decades. Figueiredo thinks she’s resourceful enough to continue uncovering new questions. “It’s as if she always has the eyes open and she’s in the world, interacting with the world,” she said.
Years after she first watched the world under a microscope, Rijo-Ferreira is still keeping time with its rhythms. In the meantime, more scientists may come to see T. brucei as she does — as something beautiful, propelling its way through a sea of red blood cells and morphing from slender to stout — something small and familiar but worthy of study, with a clock of its own.
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