A Voyage of Curiosity and Vision

ABOVE: Figueiredo’s team discovered that T. brucei parasites (in red) occupy the adipose tissue of their mammalian hosts, moving around between the extracellular spaces of adipocytes.  Mariana De Niz

Luísa Figueiredo, a parasitologist at the University of Lisbon, has a long-standing relationship with parasites. It all began during her time as an exchange student at Imperial College London when she decided to take a course in molecular parasitology. It unlocked a world of small creatures that was foreign to Figueiredo. “I was fascinated by the molecular mechanisms that parasites use to survive within a host,” she said. “That really inspired me to [pursue] a career in parasitology.” 

         

Among the helminths, ectoparasites, and protozoa that infect mammals, Figueiredo zeroed in on African trypanosomes, particularly Trypanosoma brucei (T. brucei), the causative agent of sleeping sickness, a human disease that can be fatal if left untreated.¹

By diving deep into the biology of trypanosomes and their interactions with their hosts, Figueiredo and her team have expanded researchers’ understanding of these parasites, uncovering not only the ingenious strategies that they use to evade the immune system, but also the niches that trypanosomes occupy in their mammalian hosts. 

Into the World of Parasites

Figueiredo’s journey into parasitology did not begin with single-celled trypanosomes. Instead, as a young parasite enthusiast, she moved to Paris to work with molecular parasitologist Artur Scherf at the Pasteur Institute and studied Plasmodium falciparum (P. falciparum), the most virulent Plasmodium species that causes malaria.

While in Scherf’s lab, Figueiredo focused on telomeres and the enzyme telomerase, which adds new DNA repeats to telomeres to help maintain genome integrity. In the malarial parasite, telomeres tether the chromosomes to the nuclear periphery, forming clusters that facilitate recombination among them.² Figueiredo and her colleagues found that losing a subtelomeric region affected chromosome stability in these clusters and altered the spatial organization and potentially the recombination of chromosomes.³ In another study, they identified a putative telomerase of P. falciparum and showed that the enzyme accumulates in the nucleolus.⁴ 

Genetic manipulation of P. falciparum was very challenging back then, Figueiredo recalled. So, she decided to look beyond the malaria-causing protozoan and find a parasite that would allow her to investigate the molecular mechanisms that most interested her. 

In the early 2000s, researchers had succeeded in genetically manipulating trypanosomes, flagellate protozoa best known to cause Chagas disease and sleeping sickness.⁵ The ease of gene knockout or knockdown in trypanosomes captured Figueiredo’s attention, and in 2003, she joined the laboratory of George Cross, a molecular parasitologist at the Rockefeller University whose research focused on African trypanosomes. 

T. brucei’s Cloak of Invisibility

In the early 1970s, Cross identified a family of glycoproteins in T. brucei known as variant surface glycoprotein (VSG).⁶ T. brucei has its coat surface, a structure that surrounds the parasite cell body and flagellum, densely covered with millions of copies of a type of VSG.⁷ As the parasite continues its life cycle in the bloodstream, the immune system of its mammalian host recognizes the expressed VSG and triggers an immune response to eliminate the invader. As a combat strategy, T. brucei periodically switches one VSG for another.

I was fascinated by the molecular mechanisms that parasites use to survive within a host.

 —Luísa Figueiredo, University of Lisbon

“Variant surface glycoprotein genes are critical for the parasite to evade the immune system through a mechanism of antigenic variation,” Figueiredo explained.

Figueiredo wanted to understand how the antigenic variation process works. While in Cross’ lab, she and her colleagues found that a histone methyltransferase regulates the number of VSG and the rate at which each parasite switches from one VSG coat to another.⁸ By looking at VSG genes located at a bloodstream-form expression site (BES) in T. brucei’s genome, Figueiredo also found that a transcriptionally active BES has fewer nucleosomes, uncovering changes in chromatin structure that may affect the antigenic variation in the parasite.⁹  

When Figueiredo started her own group at the University of Lisbon in 2010, she continued exploring the mechanisms regulating antigenic variation in African trypanosomes. In one of her research projects, Figueiredo’s team showed that TDP1, a high-mobility group box protein, helps keep the open chromatin structure in transcriptionally active BES in T. brucei.¹⁰ She also noted that overexpression of the TDP1 gene causes the chromatin to decondense at silent VSG loci and leads to the expression of multiple VSG, making the parasite more susceptible to the host’s immune response.¹¹   

A Fatty Attraction 

Before Filipa Rijo-Ferreira, now a molecular parasitologist at the University of California, Berkeley, joined Figueiredo’s group as a graduate student, she studied immunology and infection in the laboratory of viral immunologist Charles Bangham at Imperial College London. Rijo-Ferreira first met Figueiredo while looking for research positions when she returned to Lisbon, and although studying parasites was not on her radar, Figueiredo’s enthusiasm when talking about trypanosomes hooked her. “It became contagious. So, I ended up not wanting to work with viruses anymore, and decided to join her lab,” Rijo-Ferreira recalled. 

By the early 2010s, researchers had accumulated a vast body of knowledge about T. brucei life stage in the blood, but little was known about how the parasite affects the brain. Rijo-Ferreira found this question compelling, as people with sleeping sickness show episodes of nocturnal insomnia and patterns of daytime sleep.¹²

Given that the sleep-wake cycle is under circadian control, Rijo-Ferreira was curious whether T. brucei could affect the circadian rhythms of the host. To explore this question, Rijo-Ferreira and her colleagues set out to establish a mouse model of infection, something with which Figueiredo’s team did not have much experience. As a first step, they collected all the main organs of the infected mice to see how the infection affected those tissues. As Rijo-Ferreira harvested the organs, she recalled leaving a tiny amount of fat tissue on each organ and then sending the specimens to then University of Lisbon pathologist Tânia Carvalho, who helped the team stain the sections to identify the parasites.

A few days later, Figueiredo recalled being surprised by Carvalho’s findings. “Tânia comes into my office and says, ‘As you predicted, most organs are clean of parasites, and [there are] no signs of major pathology. However, why didn’t you tell me that all parasites accumulate in the adipose tissue?’” Figueiredo said. “She then explained, ‘Whenever I have a section that has a little bit of the adipose tissue, it’s always full of parasites.” 

“This is cool, but is it real?” Rijo-Ferreira recalled thinking when they saw the results. Over the course of months, they conducted many control experiments, varying the routes of parasite delivery and even using tsetse flies, T. brucei’s natural vector. “Every time we found parasites in the adipose tissue, and always in large amounts,” Figueiredo recalled. “That led us to conclude that this is real, at least in mice.”

This serendipitous finding drove Figueiredo to study the fat tissue, a previously unknown reservoir of T. brucei in its mammalian hosts. 

Exploring T. brucei Life in the Adipose Tissue 

Once they started looking into the fat tissue, Figueiredo and her team first investigated whether the parasites living in the adipose tissue resemble those in the blood. They found that fat tissue T. brucei residents are infective, and unlike their blood counterparts, they show upregulation of lipid metabolism and fatty acid β-oxidation genes.¹³ In vitro experiments revealed that these parasites can use a fatty acid as a carbon source, suggesting that the fat tissue dwellers functionally adapt to the adipose tissue environment.

There’s this entirely different niche that people have not known about, and it turns out to be this major reservoir for the parasites.

 —Filipa Rijo-Ferreira, University of California, Berkeley

These findings piqued the interest of Kimberly Paul, a molecular parasitologist at Clemson University, who studies fatty acid metabolism in trypanosomes. Early studies in T. brucei failed to detect fatty acid uptake and oxidation, leading researchers to believe that these parasites did not perform β-oxidation. “This became kind of dogma,” Paul said. When the T. brucei genome became public in the mid-2000s, scientists identified the genes for fatty acid oxidation.¹⁴ However, they still could not detect this metabolic process in the laboratory, probably because most experiments were done with the tsetse fly and bloodstream forms of the parasite, according to Paul. 

“[Figueiredo] was finally able to detect a form where they actually do this pathway. So, I was incredibly excited about that,” Paul said.

Although finding the parasites in the adipose tissue was a detour from Rijo-Ferreira’s plan to study T. brucei in the brain, she knew that this finding was just too exciting to ignore. “There’s this entirely different niche that people have not known about, and it turns out to be this major reservoir for the parasites,” she said. 

After their discovery of T. brucei in the adipose tissue, Figueiredo wondered what the parasite does there. Since her team knew that the fat tissue parasites, later named adipose tissue forms (ATF), metabolize fatty acids and that trypanosome infections are associated with fat loss, they hypothesized that the parasites induced changes in the lipid metabolism of the adipose tissue. 

To validate their hypothesis, Figueiredo collaborated with Rudolf Zechner, a biochemist at the University of Graz, who studies lipid metabolism and had developed a knockout mouse model of the enzyme adipocyte triglyceride lipase (ATGL), which is key to triacylglycerol breakdown (lipolysis) into free fatty acids (FFA).   

By infecting wildtype and ATGL-deficient mice with T. brucei, the researchers found that while mice lacking the ATGL gene hardly lost any fatty tissue upon infection, they had shorter lifespans and more parasites in their adipose tissues compared to wildtype animals.¹⁵ After identifying the FFA released from the adipocytes, the researchers exposed the parasites to each one of them and found that one FFA, linoleic acid, killed T. brucei in vitro. “That was in complete contradiction to what we had thought,” Figueiredo said. “We thought that the parasites would be eating those fatty acids and [that] this would be beneficial for them,” she explained.

While it is still unclear whether the lipotoxicity also happens in vivo, Figueiredo wants to keep exploring this phenomenon further and assess how factors such as the concentration of FFA as well as their spatial location in the adipose tissue might influence their effects on the parasites.   

These and other questions are not easy to address experimentally, according to Paul. “But if anyone is up to the challenge, it would be her group.”