Toxoplasma gondii is a parasite that infects hundreds of millions of people around the world. Although cases are often mild, it can cause severe symptoms in people with weakened immune systems and in developing fetuses. Toxoplasma can also persist for years by forming long-lived cysts in tissues, allowing infection to become chronic.
During chronic infection, hundreds of Toxoplasma parasites can pack into a tissue cyst inside a brain or muscle cell. That crowded life carries a cost: nutrients become harder to obtain, waste accumulates, and energy-producing reactions can become damaging. How Toxoplasma reshapes its metabolism to keep growing under such strained conditions has been unclear.
Now, a study by researchers in the lab of Whitehead Institute Member Sebastian Lourido, PhD, who is also an associate professor of biology at the Massachusetts Institute of Technology (MIT), has identified a parasite-specific protein that helps coordinate this response in Toxoplasma.
The preclinical study carried out in cells and including tests in infected mice revealed a previously unknown way that parasites regulate metabolism and identified a dedicated regulator of metabolic gene expression in apicomplexans, the group of parasites that includes Toxoplasma and the Plasmodium parasites that cause malaria.
The protein, TgPRO, allows Toxoplasma to manage oxidative stress—the buildup of reactive oxygen molecules that can damage cells—by controlling genes involved in energy production and iron use. Led by Christopher Giuliano, PhD, a former graduate student, and by graduate student Chinmay Kalluraya in the Lourido lab, the study in addition points to a possible future therapeutic strategy, indicating that inhibiting pathways controlled by TgPRO could make Toxoplasma more vulnerable to antiparasitic drugs that induce oxidative stress.
Giuliano and Kalluraya are co-lead authors of the researchers’ published paper in Cell, titled “Convergent evolution of metabolic regulation governs redox adaptation in Toxoplasma,” commenting in their report, “TgPRO is likely critical for the efficient transmission of T. gondii by enabling metabolic adaptation during chronic stages.”
Different organisms adjust metabolic gene expression during crowding, when they encounter nutrient scarcity, oxidative stress, and waste accumulation, the authors noted. “Organisms adapt to these stresses through either broad repression of biomass production or focused modulation of specific pathways.” Apicomplexans also encounter crowded environments as part of their infection cycles, the team continued. “However, while apicomplexan parasites experience these stresses during intracellular growth within host cells, they lack known regulators of metabolic adaptation.”
To discover the genes that support Toxoplasma’s ability to live in crowded cells, the researchers used a genome-wide CRISPR screen to compare Toxoplasma growing at low and high densities. The screen tests the effects of turning off genes one by one at both population densities in order to determine which genes are essential, specifically in crowded conditions. The results highlighted pathways that make or recycle NAD and NADP, molecules important for energy production and defending against oxidative damage. It also pointed to TgPRO, a previously unstudied protein that was especially important when parasites became crowded. “Nicotinamide adenine dinucleotide (NAD)(P)+ biosynthesis was required at high parasite density, along with several parasite-specific factors, including an RNA-binding protein we named ‘‘T. gondii parasite response to oxidation,’’ (TgPRO),” they stated.
“A genome-wide screen was a powerful way to ask how crowding affects parasite fitness,” Kalluraya said. “TgPRO emerged as very important at high density. Because almost nothing was known about it, we wanted to understand what it was doing.”
The researchers’ study showed that parasites lacking functional TgPRO accumulated more reactive oxygen molecules and struggled to compete at high density. Experiments showed that the loss of TgPRO disrupted the mitochondria and changed how parasites processed glucose and other nutrients. Providing additional iron or restoring an important chemical balance inside the mitochondrion improved parasite growth, connecting TgPRO’s effects to iron-dependent energy metabolism. “Collectively, TgPRO enables parasites to maintain redox balance under the metabolic strain that accompanies crowded environments,” they noted.
The team then traced the response to a molecular mechanism. TgPRO is an RNA-binding protein, attaching to the molecular messages (RNAs) that cells use to make proteins. The researchers found that it binds and stabilizes a select set of messages involved in nutrient use, mitochondrial activity, and the assembly of iron-sulfur clusters, small structures that many enzymes need to function. The experiments connected the original observation—that some parasites faltered only when crowded—to a precise interaction between a regulatory protein and its RNA targets.
“One of the really nice elements of the story is our ability to connect it all the way through—from the original observation and genome-wide screen to the metabolic consequences and the direct interaction between TgPRO and its target RNAs,” Lourido said. The researchers found that lowering oxygen levels also reduced oxidative stress and partially restored the growth of parasites without TgPRO. Toxoplasma is commonly grown in laboratories at atmospheric oxygen levels, which are considerably higher than those found in most animal tissues. The result suggests that oxygen conditions can strongly shape parasite metabolism, and the researchers caution others studying Toxoplasma to take this into consideration.
After testing the role of TgPRO in artificially crowded settings, the team also tested whether TgPRO matters during chronic infection, when Toxoplasma forms cysts in the brain. Mice infected with parasites lacking functional TgPRO developed smaller brain cysts, suggesting TgPRO supports parasite growth in the naturally dense environment of a chronic-stage cyst.
“The chronic stage is still somewhat elusive,” Giuliano said. “Showing that TgPRO affects cyst growth suggests that these same metabolic changes are needed in the brain and gives us clues about how the parasites persist there for months or years.”
TgPRO bears little resemblance to the proteins that regulate similar metabolic programs in mammals, yeast, and bacteria, yet it controls many of the same kinds of genes that these organisms adjust when cells face oxidative stress or changing nutrient conditions. This is an example of convergent evolution: distantly related organisms evolved different molecular machinery to solve a similar biological problem. That convergence suggests that coordinating these metabolic pathways may be a fundamental requirement for cells adapting to stress.
The study establishes a new paradigm for how apicomplexan parasites regulate their metabolism and advances the foundation for investigating how Toxoplasma persists inside its hosts. “Through posttranscriptional control of a coherent set of metabolic pathways, TgPRO allows T. gondii to adapt to oxidative stress, which particularly impacts chronic-stage cation,” the authors concluded. “Within apicomplexan parasites, this pathway represents a unique example of an environmentally responsive regulator driving metabolic adaptation.”
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