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Researchers characterize enzyme leading to recognition of genes involved in spread of Toxoplasma

Rima McLeod and team characterized an enzyme that plays a crucial role in the development of infectious forms of Toxoplasma gondii.

A team of researchers led by scientists from the University of Chicago have characterized an enzyme that led to genes that play a crucial role in the development of infectious forms of Toxoplasma gondii, a parasite that may affect up to a third to half of the world’s population.

This enzyme, called Toxoplasma gondii ornithine/GABA aminotransferase (TgO/GABA-AT), may lead to targets for potential vaccines and medicines and a broader understanding of conception of this parasite. In the new study, published January 19, 2024, in iScience, the scientists created a molecule that inactivates the enzyme based on the mechanism of the enzyme. The inactivator was then confirmed to bind to the enzyme by solving the structure of the enzyme with the inactivator bound. The enzyme, however, was not essential for oocyst formation. The study found this enzyme is in a newly recognized pathway for the cat stage merozoite form of the parasite.

Toxoplasma is a parasite that spreads by inadvertent ingestion of oocysts, an egg-like structure containing the parasite embryo, in food or water contaminated by cat excrement. Infection by Toxoplasma can damage eyes, compromise cognitive and motor function, contribute to epilepsies and neurodegenerative diseases, and likely sometimes malignancies, damaging the fetus and newborn infant of mothers with primary infection during gestation and causing loss of life.

“It is important to understand the early-stage life cycle of Toxoplasma to prevent its spread,” said Rima McLeod, MD, Professor of Ophthalmology and Visual Science and Pediatric Infectious Diseases at UChicago and senior author of the new study. “This organism results in lifelong infection in the brain of one-third to one-half of the human population worldwide.”

Rima McLeod, MD

Professor of Ophthalmology and Visual Science

The form of the parasite that develops after male and female forms join in the feline intestine widely contaminates food and water. An acutely infected feline excretes up to 500 million oocysts over two weeks. Even one oocyst is infectious, and they can persist in soil for up to a year or more. There are up to an estimated 400 oocysts in every square foot of soil in the United States.

In the new study, the researchers first discovered that the OAT enzyme is present in the sporozoite in the oocyst stages that causes infections in humans rather than the tachyzoite and bradyzoite stages causing disease in humans and is also present in some of the cat intestinal pre-oocyst stages. Although it is not essential for the cat to produce oocysts, instead, this enzyme is regulated by a newly described plant-like collection of proteins that control the development of the cat stage of the parasite, called merozoites. This observation led to further sequencing and bioinformatics studies performed by Hernan Lorenzi at the J. Craig Venter Institute with Dr. McLeod. These studies were performed to understand some of the gene products important in conception of the parasite in the cat intestine.

Many decades ago, a clone of Toxoplasma was created that could not form oocysts in cats and prevented infection of livestock animals in the vicinity of acutely infected cats as well. It had many mutations, however, and was difficult to produce even though it was effective as a vaccine. In the current study, the team sequenced the genes of the mutated parasite clone and compared that sequence to the sequences of the same genes in the parental clone and a collection of parasites from natural infections. They saw that the natural isolates would not be likely to survive in natural infections if they were mutated. Examination of the intestine of cats infected with this vaccine strain showed that the male and female forms of the parasite produced also did not fuse. Establishing which genes with mutations were expressed in merozoites was important in down selection of genes based on this understanding of the biology of the mutant.

Surprisingly, the candidate mutations were all in the merozoite and not in the male or female gametes. This demonstrates that the steps necessary for the conception of the parasite in the cat intestine that were abnormal in the mutant strain were in the formation of the gametes from the merozoite stage.

The proteins encoded by Toxoplasma genes critical for fertility were homologous to genes important for fertility in other species. These were further characterized using AlphaFold2, an artificial intelligence program that predicts protein structures. Ekaterina Filippova, a research professional at UChicago, placed the mutations on the protein structures of the candidate gene sequences potentially contributing to the deficiencies in parasite oocyst formation, resulting in a short list of mutations that could affect conception of Toxoplasma during capacitation of the pre-gamete merozoite stage. A larger list of mutations also provides a useful tool to understand genes essential for capacitation of merozoites and formation of the embryo of Toxoplasma.

“This could provide a foundation and a method to develop a vaccine or medicines for domestic cats to prevent contamination of the environment, since preventing oocyst shedding by cats is foundational to blocking transmission of the parasite,” McLeod said.

Researchers who contributed to this work include: from UChicago (Rima McLeod, Ekaterina Fillipova, Joseph Lykins, Ying Zhou, Kamal El Bissati, Cong Hua, Kelsey Wheeler, and Sarah Dovgin,); Northwestern (Richard Silverman, Matthew Moschitto, Hoang Le, Kristin Flores, and Wayne Anderson ); J Craig Venter Institute/NIH (Hernan Lorenzi, Seesandra Rajagopala); USDA (Jitender Dubey); University of Tennesee (Chunlei Su); Dartmouth University (David Bzik, Barbara Fox); Instituto Superiore di Sanita Rome (Furio Spano); Albert Einstein College of Medicine (Louis Weiss, Tadakimi Tomita); Leeds University (Martin McPhillie, Colin Fishwick, Stephen Muench); Strathclyde University (Craig Roberts), and Walter Reed Army Institute of Research (Mark Hickman, Patricia Lee).

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