What makes for a “skilled” parasite? According to Clemson University College of Science assistant professor of biological sciences Zhicheng Dou, high transmissibility but slow host mortality are two characteristics that make parasites successful. A parasitic disease can't spread if it kills its host before infecting someone else.

Unfortunately for Toxoplasma gondii, the parasitic cause of the disease toxoplasmosis, new research conducted by Dou has the potential to annihilate the single-celled parasite in the cyst stage of its life cycle. The findings -- recently published in Nature Microbiology -- 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 U.S. 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.

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.

However, if the host becomes immunocompromised — whether from cancer, old age or 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.

Dou began this inquiry 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 (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.

“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 becomes blocked, which causes a buildup of autophagosomes in the parasite’s cytoplasm that quickens the rate of starvation for the cysts, thus triggering their death.

Dou’s study presented these results in culture, which mimicked the conditions inside 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 VAC to the parasite’s heme synthesis pathway.