CLEMSON, South Carolina — What makes for a “skilled” parasite?

If you asked Zhicheng Dou, an assistant professor of biological sciences in the Clemson University College of Science, he would tell you that high transmissibility, yet slow host mortality are two characteristics that make parasites successful. After all, how can a parasitic disease spread if it kills its host before infecting someone else?

Unfortunately for Toxoplasma gondii, the parasitic namesake for a disease called toxoplasmosis, new research conducted by Dou has the potential to annihilate the single-celled parasite in the cyst stage of its life cycle. As recently published in Nature Microbiology, the findings suggest that the parasite’s “stomach” is a key target for drug development against toxoplasmosis.

Worldwide, nearly 2 billion people are infected with T. gondii — 60 million of whom reside in the United States. The disease itself is not contagious, except in instances of congenital transmission where a newly infected pregnant mother passes the infection on to her unborn child. Otherwise, the parasite is contracted from consuming undercooked, contaminated meat or coming in contact with cat feces containing the oocyst form of toxoplasma. This is why pregnant women are advised against cleaning their cats’ litter boxes.

Zhicheng Dou studies toxoplasmosis as an assistant professor in the department of biological sciences.

Zhicheng Dou studies toxoplasmosis as an assistant professor in the department of biological sciences.
Image Credit: Hannah Halusker / Clemson University

During the acute stage of infection — the first two to three weeks after T. gondii is introduced — some people report experiencing mild flu-like symptoms. At this point, the parasites are quickly replicating throughout the circulatory system in a form called tachyzoites. When the tachyzoites cross the blood-brain barrier — a membrane structure that typically blocks viruses, parasites and bacteria from reaching the brain — the infection shifts to its chronic stage.

“After the toxoplasma get in the brain, they don’t have access to many nutrients. This lowers their metabolism, so they start to grow slowly,” Dou said. “We call these bradyzoites — ‘brady’ means slow. In the brain, they just kind of hibernate to hide from the host’s immune system. The bradyzoites become protected inside of cyst structures, where the cyst wall is very tough to break. The difficult part is that there is no way to treat this chronic stage because no drug can cross the blood-brain barrier to control the infection.”

Without drug accessibility, the bradyzoites are able to take root in the host’s brain, where they can grow and replicate for as long as the host is alive. In and of itself, this isn’t detrimental to the host, because most people have competent immune systems that can keep the infection at bay. Even people with weakened immunity can take antibiotics, like pyrimethamine or sulfadiazine, which will suppress the infection.

“But if the host becomes immunocompromised — if they get cancer, become old or have to get an organ transplant — it will wake up the parasites in the brain and they’ll go back to the circulatory system, causing recurrent infection,” Dou said.

Thus, the path toward developing a therapeutic drug treatment for toxoplasmosis lies in obliterating the cyst form of T. gondii in the brain before the parasites can repopulate the circulatory system.

“If a drug can cross the barrier without compromising its efficacy, that would be fantastic. You could totally and completely eradicate the bradyzoites,” Dou said.

This inquiry is one that Dou began as a postdoctoral researcher at the University of Michigan Medical School in 2010. Seven years later, the results are showing that a specific enzyme — cathepsin protease L, or CPL — directs the digestive function of T. gondii’s “stomach”: the vacuolar compartment (VAC). By deleting the CPL gene from the parasite’s DNA, the toxoplasma could no longer perform an energy-saving process called autophagy.

Bradyzoites (blue) are the cyst-form of the toxoplasma parasite that reside in the brain. Autophagosomes (pink) are digested by the parasite's VAC energy-saving process called autophagy.

Bradyzoites (blue) are the cyst form of T. gondii that reside in the brain. Autophagosomes (pink) are digested by the parasite’s VAC in an energy-saving process called autophagy.
Image Credit: Carruthers Lab / University of Michigan Medical School

“Autophagy means ‘to eat self.’ During a stressed situation, like when T. gondii cannot get enough food, they have to make autophagosomes so they can rescue themselves for longer survival,” Dou said.

The autophagosomes are membranes within toxoplasma that envelop cellular contents, like proteins or other damaged organs. Once enveloped, the autophagosomes are digested by the VAC, providing energy to the parasite to continue its survival; much like how the human body begins to consume muscle under nutritionally deprived conditions, the process of autophagy in toxoplasma is a self-preservation tactic.

Without CPL, autophagy was blocked, which caused a buildup of autophagosomes in the parasite’s cytoplasm, the part of the cell that holds all of its organs. This buildup quickened the rate of starvation for the cysts, triggering their death.

Dou’s study presented these results in culture, which mimicked the conditions inside of a host, but also in mouse models. When mice were infected with mutant T. gondii that lacked the CPL gene, approximately 100-fold fewer cysts formed after five weeks than in mice infected with normal T. gondii. This rate increased 100-fold more by 16 weeks post-infection. These results link CPL and the stomach it controls (the VAC) as possible targets for treating the chronic stage of toxoplasmosis.

As part of their study, Dou and his Michigan collaborators tested a potential CPL inhibitor (LHVS) and found that the drug was able to penetrate the cyst wall, gaining entry to kill the bradyzoites. However, LHVS is not able to cross the blood-brain barrier, making it currently inaccessible to the cysts once administered to the host.

“This research is important for two reasons,” Dou said. “First, it gives a proof of concept. This organelle, the vacuolar compartment, is a therapeutic target for drug development. If you interfere with the stomach, the parasite cannot digest food or eat itself, so it cannot survive. Secondly, my former lab is now trying to modify the drug (LHVS) by collaborating with chemists so that, hopefully, it will one day be able to cross the blood brain barrier.”

Since coming to Clemson University in 2015 and beginning his own research lab, Dou has shifted his target of interest for a therapeutic toxoplasmosis treatment from the vacuolar compartment to the parasite’s heme synthesis pathway. Heme is an essential prosthetic nutrient for all organisms. Although Toxoplasma can synthesize heme on their own, Dou’s latest findings suggest that the parasite can also scavenge heme or heme intermediates from host cells as a supplementary source. Disruption of heme biosynthesis in Toxoplasma causes dramatic growth and virulence defects, indicating its therapeutic potential to combat toxoplasmosis.

“It takes a while to conduct toxoplasmosis research because it’s tough to do. But it is worth it, and it’s fun to study the pathogenesis of toxoplasmosis,” said Dou, who is hopeful that his collaborative research will result in a successful treatment in the future. “It will not only help identify new therapeutic strategies, but also reveal the exciting interaction between host and microbes during evolution.”


Dou’s publication — titled “Toxoplasma depends on lysosomal consumption of autophagosomes for persistent infection” — was published in June 2017 in the second volume of Nature Microbiology. The study was supported by the National Institutes of Health under grant numbers R01AI120627 and R01AI060767. Dou received additional support from the American Heart Association for his fellowship as a postdoctoral researcher. The researchers are wholly responsible for the content of this study, of which the funder had no input.