Bioluminescent parasite is used to study chronic-stage Chagas disease

Technique developed at London School of Hygiene and Tropical Medicine also helps determine efficacy of anti-Chagas drugs.

By Karina Toledo

Agência FAPESP – Researchers at the London School of Hygiene & Tropical Medicine (LSHTM, UK) have used the red light emitted by a genetically modified version of the Trypanosoma cruzi parasite that causes Chagas disease to monitor even very low levels of infection in mice.

Originally developed to help understand chronic-stage Chagas disease, the technique has proved to be a powerful tool for gauging the effectiveness of anti-Chagas drugs.

The method was presented in June by one of its creators, John Kelly, a professor of molecular biology, during the São Paulo School of Advanced Science on Neglected Diseases Drug Discovery – Focus on Kinetoplastids (SPSAS-ND3).

The event took place at the National Bioscience Laboratory (LNBio) in Campinas (São Paulo State, Brazil) and was supported by FAPESP.

“It’s been hard to study the disease in its chronic stage, as the T. cruzi parasite load in the organism is too small to be detected by traditional methods. The detection range for this new technique is between 100 and 100,000 parasites, which is highly sensitive. Theoretically, we can identify infection even if it’s confined to a single cell,” Kelly told Agência FAPESP.

Generally transmitted by a bite of the Triatoma infestans bug or contaminated food, Chagas disease begins with an acute phase which may be asymptomatic or produce symptoms such as fever, fatigue, nausea, swollen glands, rash, enlargement of the liver and spleen, and swelling of the eyelids (Romaña’s sign).

Chronic-stage complications, which may appear years later, include ventricular enlargement (affecting approximately 30% of patients and typically leading to heart failure) and dilation of the esophagus and/or colon (the latter affecting up to 10% and potentially leading to paralysis of peristaltic bowel movements and sphincter dysfunction).

“One of the puzzles scientists have tried to solve is why some people develop severe symptoms in the chronic stage of the disease, while others are practically asymptomatic,” Kelly said. “What we do know is that infection by T. cruzi is for life in every single case. Somehow the parasite manages to avoid being completely eliminated by the immune system. It clings on in a few cells, and proliferation resumes at times of immunodeficiency.”

To understand more about how this happens, the researchers at the LSHTM infected mice with a version of the parasite that had been genetically modified to express the gene for the enzyme luciferase, which comes from the firefly.

Their key innovation, however, was modifying the enzyme so that the reaction catalyzed by it produced red light, which is more easily absorbed by body tissue and hence enhances the sensitivity of the technique.

The luciferase gene was supplied by Bruce Branchini, a researcher at Connecticut College in the United States.

“When luciferase reacts with the protein luciferin, which is its substrate, it normally causes emission of yellow-green light. But most of the light that penetrates tissue is absorbed by hemoglobin, which is red. In other words, hemoglobin absorbs all colors except red,” Kelly explained.

Moreover, shorter wavelengths, such as those of blue and green light, are dispersed more rapidly than those of red light, which are longer.

“These two factors account for the increased sensitivity of the method using red-light emission,” Kelly said.

First experiments

The mice infected with the modified parasite were monitored for 377 days. The disease enters its chronic stage after the 70th day.

“Initially, we performed a series of tests to be sure that genetic modification wouldn’t have an effect on the parasite’s capacity to infect cells or reproduce, as this could bias the results,” Kelly said.

When luciferin is injected into mice, it is oxidized by luciferase, and the red light that is then emitted is captured using an IVIS Spectrum in vivo imaging system. The images generated can be used to measure parasite load in each stage of infection and to identify the cells and organs in which parasites are concentrated.

“Using this method, we discovered that the parasite hides in the intestine during the chronic stage,” Kelly said. “For some reason, the immune system is able to clear T. cruzi from all organs except the intestine. We’re now trying to understand why.”

When immunodeficiency was induced via administration of the chemotherapy drug cyclophosphamide, the parasite load increased again and other organs began exhibiting signs of infection.

“Traditional methods can’t detect the parasite during the chronic stage of the disease, so until now it’s been impossible to determine whether a particular drug was really capable of curing it. In this sense, our discovery represents genuine progress,” Kelly said.

In one of the experiments, the researchers compared the effects of benznidazole, the main drug used to treat chronic Chagas today, with those of posaconazole, which was considered a new hope for patients but has not been successful in initial clinical trials.

“We treated the infected mice with posaconazole and observed that the parasite load appeared to have been eradicated. But, when we induced immunosuppression with cyclophosphamide, the infection returned. So, in fact, the animals hadn’t been cured,” Kelly said.

The same experiment was performed using benznidazole, and following immunosuppression even intestinal infection had been eradicated. “The result suggests that benznidazole is genuinely effective to control chronic infection,” he stressed.

According to Kelly, the bioluminescence technique can also be used to investigate the mechanisms of infection by other species of Trypanosoma and by parasites of the genus Leishmania.