Parasitic infections pose challenges to creating effective therapies because these microorganisms are eukaryotic, like the humans and animals that they infect. Additionally, species like Toxoplasma gondii live inside cells, making studying them more difficult and adding to the troubles of developing treatments.
“In order to [discover] better targets, we need to explore in detail the changes that this parasite is inducing in the host cells,” said Gina Gallego-López, a postdoctoral researcher and parasitologist at the Morgridge Institute for Research and University of Wisconsin-Madison. Many of the amino acids and lipids that T. gondii requires for its survival are products of host metabolism, therefore, understanding how the parasite manipulates host metabolic pathways may suggest new strategies for defeating this invader.1
Recently, Gallego-López and her colleagues developed a new approach for studying intracellular infections in which they applied two-photon microscopy to noninvasively measure metabolic changes in T. gondii-infected cells.2 This research, published in mBio, demonstrated that T. gondii altered metabolic activity in human fibroblasts, shedding light on parasite-induced metabolic rewiring.
“It’s really hard to separate host cell metabolism from parasite metabolism,” said Laura Knoll, a parasitologist at the University of Wisconsin-Madison and study coauthor. To overcome this, the team used T. gondii that expressed the red fluorophore mCherry to track the locations of the parasites. Then, they leveraged natural fluorescence from cellular respiration metabolites to monitor changes in the amount of these products during infection.
The team began their studies with a less virulent strain of T. gondii that proliferated more slowly which allowed them to study changes in metabolism over time. They gained insights into cell metabolism by measuring nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, together called NAD(P)H, two metabolites that accept or donate electrons during cellular respiration. Thus, changes in the proportion of NAD(P)H bound to proteins versus free enzyme served as a reliable indicator of cellular metabolism. They observed that T. gondii infection increased the ratio of bound enzymes and created a more oxidized intracellular environment, both of which indicate increased metabolism.
Using two-photon microscopy, the team tracked the amount of NAD(P)H in host cells over the course of infection by capturing the metabolites’ autofluorescence. The concentration of NAD(P)H is inferred by the color gradient, with blue being less metabolite and red being more.
Gina Gallego-López
“We didn’t have any idea about redox changes induced by Toxoplasma gondii in the host,” said Gallego-López. “This is the first time that someone was able to measure that in alive cells.”
To further explore the metabolic effects of T. gondii infection, the team studied changes in the concentration of glucose and lactate. They showed that glucose gradually increased in the first nine hours after infection, then decreased over the remaining 48 hours. Meanwhile, the cellular lactate concentration began decreasing after six hours of infection. This corresponded with increased glycolysis early in infection and then decreased glycolysis after 24 hours, paralleling the greater intracellular oxidation, suggesting that the parasite alters glucose metabolism for its growth.
The team compared these findings to their previous RNA sequencing results.3 They found that the increased gene expression they previously observed in host cells corresponded to enzymes involved in cellular respiration. Additionally, they observed increased expression of reactive oxygen species-metabolizing enzymes in T. gondii as well as other metabolically important enzymes.
The present study and its agreement with the team’s prior gene expression data indicates that T. gondii alters cellular metabolism. Knoll’s team suspects this could provide clues about viable therapeutic strategies. The group previously demonstrated that a cancer drug candidate reduced T. gondii growth in cells.4 “Cancer is rapidly replicating eukaryotic cells that avoid the immune response, and that’s what [intracellular parasites] are as well,” Knoll said. “So, we should rethink the drugs we already have, and some of those might be very useful drugs [against T. gondii].”
Zhicheng Dou, a molecular geneticist who studies T. gondii at Clemson University and was not involved with the study, said that the work was interesting, and that the imaging technique would be useful in the field once more labs applied it and demonstrated its validity in more models. He is also interested in more exploration into the mechanisms behind the altered metabolism.
“This is something probably we should understand in the future,” Dou said, adding that the field can explore where these changes are coming from. “The host cells may change their metabolism to resist the infection.”
