A group of Brazilian researchers worked together in 2010 at the University of Maryland in the U.S. to conclude a study that detailed the strategies that the protozoan Trypanosoma cruzi adopts to invade and occupy human cells. This is the first step that triggers the infection that characterizes Chagas Disease, still relatively common in some South American countries. The researchers’ conclusion indicates that the Trypanosoma cruzi makes cell repair mechanisms work for its benefit. This helps explain the parasite’s affinity with muscle cells that cause the heart to expand, which is one of the characteristics of the disease’s chronic phase. The disease’s mortality rate has dropped in the last few years, but estimates are that from 3 to 5 million people a year still suffer from chronic Chagas Disease.
Physician Carlos Chagas had already seen the T. cruzi in the blood and cells of infected patients by using a microscope in his lab at Rio de Janeiro’s Oswaldo Cruz Institute. In the 1940’s, Herta Meyer, of the Federal University of Rio de Janeiro (UFRJ), made a film showing how the parasite invades the cells and reproduces inside them (the film is available on the web page of the Brazilian Protozoology Society – enter the site and then click on interage and multimedia). This was part of the pioneering work of these researchers, who first identified the agent that causes the disease, the transmitting insect and the characteristic symptoms. The limitations of their equipment did not allow these researchers to go much beyond their initial findings (see Pesquisa FAPESP nº 163). In Maryland, however, equipment and researchers with converging interests were available.
Biologist Maria Cecilia Fernandes, who was enrolled in the doctorate program at the Federal University of São Paulo (Unifesp), had already spent two years in Maryland studying the mechanisms through which the T. cruzi invades human cells. She was working at the cell biology lab, under the coordination of Norma Andrews. Norma has investigated the interaction between the T. cruzi and the host cells since the 1980s, when she was studying for her doctorate degree at the University of São Paulo (USP) under physician and biochemist Walter Colli, one of Brazil’s leading experts on Chagas Disease. One of her lines of research focuses on the repair mechanisms of the cell membrane. In April 2010, Renato Mortara, a professor at Unifesp who has studied the behavior of T. cruzi since the 1980’s, joined the group in Maryland.
Step by step
As the researchers needed other kinds of equipment, they asked two Brazilian researchers who were at Bethesda – which lies less than an hour away from College Park – to help them. The Brazilian researchers were Bechara Kachar, a researcher with a medical degree from the University of São Paulo (USP), who has been working at the National Institute of Health (NIH) since 1986, and Leonardo Rodrigues de Andrade, a researcher from the Federal University of Rio de Janeiro (UFRJ) who is currently working at Kachar’s laboratory.
With the help of colleagues from the USA, the researchers studied, filmed and arrived , by means of proof and counterproof, at the step-by-step process encompassing the invasion and occupation of the cell by the T. cruzi. Probably because the parasite moves around so much, it damages the cell’s outer membrane until a tiny hole is made, This hole is the entry for calcium ions, which are plentiful in the space outside the cell, as the group headed by Norma had already described. Other research groups had also extensively studied the behavior of the T. cruzi. For example, some years ago, Sergio Schenkman and his team from Unifesp had described how the parasite escaped from the defense mechanisms of the organism it invades (see Pesquisa FAPESP nº 118).
This new study shows that, inside the cell, the ionic calcium activates the merger of compartments called lysosomes with the outer membrane that surrounds the tiny orifice made by the parasite. When merging with the membrane, the lysosomes release an enzyme called sphingomyelinase, which leads to the formation of ceramide, another molecule of the membrane. The ceramide, in turn, forms a curve on the membrane, removes the injured part, and repairs the membrane, thus facilitating the parasite’s entry.
“The parasite benefits from the enzyme and the ceramide – the cell repair mechanism in the membrane,” says Mortara. “The T. cruzi is attracted to muscle cells, which are more prone to injuries and this is why they activate the outer membrane’s repair mechanisms more frequently.”
Inside the cell, the protozoa moves actively in compartments that are similar to the lysosomes. “The intense movement probably attracts other parasites to the same cell,” Mortara adds. The T. Cruzi releases toxins that allow it to escape. The parasite starts to multiply and can generate a hundred copies a few days later. The overpopulated cell stops functioning and breaks open, releasing parasites that colonize other cells.
Like an airplane pilot approaching a city, the micro organism needs to pinpoint exactly where to touch a cell. Of course the parasite has no intention of landing, but it must find an electrostatic affinity with some part of the cell – if it makes the wrong choice, this mistake can trigger defense mechanisms that tend to eliminate the undesired visitors. Bacteria, viruses, and protozoa adopt different strategies that allow them to cross the membrane and invade a site where they might find some protection to then reproduce. “The protozoa that cause leishmaniasis seem to exploit a cell repair mechanism, that is similar to that of the T. cruzi, to invade the macrophages,” Mortara explains.
Each micro organism survives by means of specific ways of making cells work in their favor. In the August issue of Cellular Microbiology, Rey Carabeo, from London’s Imperial College London, described various invasion strategies adopted by bacteria that cause problems to human beings, such as the Salmonella, the Escherichia coli and the Chlamydia. In general, these bacteria activate the mechanisms that produce a remodeling of actins, the abundant proteins found just below the cell membrane. Each micro organism, when touching the host cell, activates different proteins that bind with the actins, inducing the formation of elongated pillar-like molecules that stretch the cell. This results in extensions of the cell surface that engulf the micro organism and allow it to invade the cell without injuring the membrane. In the July issue of Molecular Microbiology, James Bamburg, of the US’s Colorado State University, described in detail the chaining of molecules that facilitate the entry of the Listeria monocytogenes, a bacterium that can cause severe infections, especially in pregnant women.
Chagas Disease no longer causes much concern. The battle against the transmitting insects has resulted in a decline in the number of contaminated people. Public health authorities registered less than 200 cases and only three deaths from this disease in 2008. Although there are fewer houses built of wattle and daub with holes where the insects hide, there is still reason for concern. Contamination is still an issue because of poor hygiene and lack of attention. The disease can also result from the consumption of contaminated sugarcane juice or açai juice, and – more rarely – by contamination through blood transfusion or organ transplants.
At a conference in 2010, Norma commented that this is a disease that is associated with poverty. There are approximately 18 million contaminated people in Central and South America. The disease is not easily detected: 41% of the people that carry the T. cruzi do not develop any symptoms of the disease. The heart expands in 45% of the contaminated patients and the stomach or the esophagus dilate in 11%. Norma states that it will be difficult to eradicate Chagas Disease entirely, because the parasite can be transmitted to human beings by at least 100 species of mammals, including dogs, cats, and rodents. Because these animals usually live near peoples’ homes, they become reservoirs for the parasite after their blood is sucked by the transmitting insects.
Molecular studies of the Trypanosoma cruzi and its interaction with cells and host factors in vitro and in vivo (nº 2006/61450-0); Modality Thematic Projects; Coordinator José Franco da Silveira Filho – Unifesp; Investment R$ 1,523,719.55 (Fapesp)
FERNANDES, M.C. et al. Trypanosoma cruzi subverts the sphingomyelinase-mediated plasma membrane repair pathway for cell invasion. The Journal of Experimental Medicine. v. 208, n. 5, p. 909-21. May 9, 2011.