No kittens required: Scientists find new way to study toxoplasmosis parasite in lab

One of the most widespread parasites on the planet can also be one of the most difficult to study. Toxoplasma gondii —a single-celled protozoan—is capable of infecting almost every mammal and bird species, including humans , and in severe cases causes blindness, birth defects, and death. Yet it only sexually reproduces inside the intestines of domestic cats and other members of the Felidae family, prompting controversial studies on kittens.

Now, a research team has come up with a technique that uses gene editing of Toxoplasma to push the parasite toward sexual development in cells grown in the lab. The success is shining new light on this mysterious phase of the parasite’s life cycle—no felines required.

“It’s fantastic work,” says Alex Rosenberg, a microbiologist at the University of Georgia, Athens, who was not involved in the research. “Previously we couldn’t really look at those stages of the parasite because they’re not easily accessible. … Even if you have a cat, it’s not that easy to study.” The in vitro technique, he adds, “opens a new field for us.”

Toxoplasma ’s life cycle involves both an asexual phase—comprising the stages that cause disease—and a sexual phase, in feline intestines. Asexual versions of Toxoplasma are relatively easy to grow in the lab. But sexual forms are not, meaning scientists have struggled to gain a full picture of how the parasite multiplies and gives rise to the various different strains infecting people around the world.

Researchers used to feed kittens meat laden with Toxoplasma and harvest the sporelike parasite particles shed in the animals’ feces. Those cats were then euthanized, sometimes just a few weeks later, and incinerated to avoid spreading the infection.

The practice has long raised ethical concerns and drawn criticism from animal welfare activists. In 2019, the United States’s most prominent Toxoplasma lab shut down its work after coming under pressure for infecting and euthanizing dozens of cats each year. Very few labs still use felines, and the search is on for a better alternative.

One attempt came a few years ago from a team led by Laura Knoll at the University of Wisconsin, Madison. The researchers treated mice with drugs and dietary supplements to make the animals’ intestines more like those of felines, and successfully pushed Toxoplasma from its asexual phase into sexual development . But the approach was expensive and not very practical, Knoll says. Her group is still tweaking the technique.

In the new study, posted as a preprint in January, Mohamed-Ali Hakimi and colleagues at Grenoble Alpes University tried a different tack. They knew that shortly after infecting a cat, asexual Toxoplasma starts making presexual cells known as merozoites, which in turn go on to form macro- and microgametes—the parasite version of egg and sperm cells.

They also knew that Toxoplasma moves from one life stage to another by switching on and off various genes. If researchers could replicate these changes in the lab, Hakimi reasoned, they’d be able to take an asexual parasite and force it to form a merozoite, even outside of a cat.

Using the gene-editing technology CRISPR in Toxoplasma , the researchers began systematically disabling the genes for proteins known as AP2 transcription factors, which influence gene activity during the parasite’s development. Simultaneously disabling two of those factors, AP2XII-1 and AP2XI-2, had just the effect the team wanted: it made the parasite switch off genes associated with its asexual phase, and switch on genes usually active in merozoites.

“The data is just beautiful,” Knoll says. She praises the amount of molecular detail in the team’s study, highlighting several experiments the researchers did to show how the AP2 transcription factors interact with other proteins and the parasite’s DNA to regulate gene activity. “It’s very nice, precise biochemistry.”

Lena Pernas, a microbiologist at the Max Planck Institute for Biology of Ageing, is among those excited by the advance. “There’s a lot of really cool cell biology questions we could approach by studying the merozoite life stage,” she says, not to mention broader questions about Toxoplasma biology, like why cats are the parasite’s principal hosts.

For now, the team’s technique only replicates the earlier stages of Toxoplasma ’s sexual development. The labmade merozoites in the study didn’t go on to make mature micro- and macrogametes, meaning no sex. “There’s something missing that they’re not able to get it farther,” Knoll notes.

It’s possible that proper gamete formation only happens in the presence of some specific ingredient of the cat gut, Hakimi says. Or perhaps scientists could manipulate additional transcription factors to trigger this step—something his group is exploring now, he adds.

Such research could do more than just spare cat lives. Hakimi, who has already made the team’s gene-edited Toxoplasma strains available to other research groups, says better knowledge of Toxoplasma development could help scientists identify drugs that halt or accelerate different stages of the parasite’s life cycle, and perhaps block it from causing disease.

Getting Toxoplasma to reproduce sexually in the lab would also open the door to so-called classical genetics experiments, such as crossbreeding different strains of Toxoplasma . Those could help researchers identify genes for virulence and other disease-related traits . “That would be amazing,” Knoll says. “This is definitely getting us another step further.”