from Paris and São Paulo
On the morning of April 2 this year Brazilian biodoctor, Rogerio Amino, crossed Rue Dr. Roux and walked quickly towards a modern building in the 15th arrondissement, a middle class neighborhood in Paris. He went through the security door and along a maze of corridors until he entered a dark room where two researchers were operating a laser scanning confocal microscope that allows three dimensional images of live tissue to be observed and reconstructed. On a screen similar to that of a computer they were following the movement of small green fluorescent balls. They were specimens of the parasite that causes malaria that were being developed on skin cells. In a rapid chat Amino saw that the experiment was proceeding as planned and went back to his own laboratory on the other side of the street, less than 300 meters away. The group, which the Brazilian biodoctor has belonged to since January as a researcher, hired by the Malaria Biology and Genetics Unit of the Pasteur Institute in Paris, and which is headed up by French doctor Robert Ménard, has been paying twice as much attention to its work than normal and repeating each step of the experiment.
The team knew it was looking at an important discovery in the life cycle of the plasmodium parasite, a one-celled organism that every year infects around 250 million people worldwide, especially in Africa, Asia and Latin America, and kills almost 1 million, most of them children under 5.
The new aspect being pursued by Amino and a part of Ménard’s team is that in the organism of mice – and possibly in humans – the malaria parasite does not develop and matures exclusively in liver cells, where in two days each protozoan generates thousands of copies capable of invading the red blood cells, causing the shivering, fever and intense muscle pain that is typical of malaria. When they are injected into the skin by the bite of female Anopheles mosquitoes some specimens of plasmodium remain there where they reproduce and reach the stage when they become capable of penetrating the blood cells.
The discovery, which was presented in May at the Pasteur Institute in a seminar for European community researchers and that is about to be published in an international scientific journal, is far from representing a cure for malaria, a disease that has possibly accompanied the human species since it first appeared in Africa 200,000 years ago. But identifying this hitherto unimagined phase in the protozoan cycle should contribute to the search for more efficient ways of combating it. The compounds used for eliminating plasmodium, like chloroquinine or the artemisinins, only act in the blood phase of the infestation, in which a single protozoan generates dozens of copies every 24 hours inside the red blood cells. “Even primaquine, a compound capable of eliminating parasites in the liver, does not attack those that develop on the skin”, explains Amino. So the same organism that keeps mammals in contact with the environment and protects them from contamination may function as a preserve for malaria parasites. “We need to find a way of attacking them there”, he says.
The number of protozoans that settle on the skin, where they may remain alive for weeks, is not small. In experiments carried out at the Pasteur Institute female Anopheles stephensi mosquitoes, which transmit human malaria in Asia, were fed on the ear of the mice. In the bite the insects injected dozens of parasites marked with a green fluorescent protein into the animals’ skin. Around 10% of the protozoans of the Plasmodium berghei species, which causes the disease in rodents, lodged a few millimeters from the bite, while some migrated to the hair follicles, a structure that surrounds the hair and that has no active immune response. “There are few such privileged places for a parasite to survive in the organism of mammals”, says Amino.
These protozoans remained in the follicles for at least two weeks – it is not known if they were dormant or growing slowly. When extracted from the follicles they were capable of infecting skin cells in vitro and reaching the stage at which they invade the red globules. When the researchers injected them into healthy mice the rodents became sick. Now the group is trying to repeat the tests with mice that have received a human skin implant – which would allow work with protozoan species that contaminate people – to see if the development on the skin is the same in human beings.
Before these experiments not much attention was paid to what happened on the skin. It used to be believed that the forms of protozoan inoculated in the mosquito bite reached the blood stream and migrated to the liver where they matured before returning to the blood. Working over the last few years in Ménard’s team, Amino has been showing that it is not that simple. Indeed, this is not the first dogma on the biology of the plasmodium that the Pasteur group has broken over the last four years, thereby maintaining the pioneering tradition of the institution that was created by chemist, Louis Pasteur, and by physician, Pierre Émile Roux, 120 years ago.
During the previous period when he worked at the Pasteur Institute, Amino and German parapsychologist, Friedrich Frischknecht, had discovered that the mosquito did not inject the protozoans directly into the blood vessels, as was believed. Instead, some dozens of parasites were deposited in a deep layer of the skin where they moved in a circular fashion, like the movement of a corkscrew, to the nearest blood vessel, they described in an article in Nature Medicine (see Pesquisa FAPESP nº 120) in 2006. “People take what is not said as having been said. It’s very easy to extrapolate an idea that makes sense from the logical point of view for which there is no experimental proof or direct evidence”, Amino states.
In that same year Amino and Ménard, in partnership with Germans from the Bernhard Nocht Institute of Tropical Medicine, revealed details of how the plasmodium matures in the liver cells (hepatocytes), which helped them understand why the organism has difficulty in identifying the plasmodium as an invader and eliminating it. It was imagined that once installed in the hepatocytes some days after the bite, the protozoans multiplied and generated thousands of copies until the host cell exploded, liberating the parasites into the blood. Experiments with the rodents revealed a very much more subtle and effective strategy.
As they multiplied and matured the parasites were gradually eliminated from the hepatocytes into the vesicles formed by the cell lining of the host itself. In an article in Science, the researchers nicknamed it the Trojan hepatocyte strategy, a reference to the ruse used by the Greeks in the Trojan War. In this cunning flight the parasites also avoided the dying hepatocyte signaling to the defense system that it was time to move into action. “This protection allows us to understand why the parasites escaped the immune system cells in the liver”, explains Amino.
It is not just at the Pasteur Institute that new things are appearing. In Brazilian investigation centers in São Paulo, Rio de Janeiro, Minas Gerais, Acre and other states researchers are also working on revealing the life cycle of the plasmodium and the behavior of the insects that transmit it.
At the Biosciences Institute (IB) at the University of São Paulo (USP), the team of biochemist, Celia Garcia, is investigating what happens with the plasmodium after it gets into the red blood cells. Over the past ten years Celia and her helpers have identified at least four important mechanisms that explain how the parasite survives and matures inside the red blood cells, an environment that ought to be extremely inhospitable to it.
The concentration of calcium in the red blood cells is 10,000 times less than in the plasma, the medium in which they are diluted. The lack of this chemical element that regulates different cell functions – among them programmed death – should stop the protozoan from lodging in the red blood cells, but it does not. Celia and Marcos Gazarini observed that the secret of survival in this environment is the way the red blood cells are invaded. Instead of opening a small hole in the blood cell’s membrane the parasite pushes into the membrane, thus forming a pocket around itself. Inside this pocket the calcium level is the same as that of blood plasma.
In collaboration with pharmacologist, Regina Markus, also from USP, Celia managed to unveil another aspect of the curious behavior of the plasmodium. Once inside the red blood cell the parasite multiplies and goes through three different development phases. At the end of this period of transformation, which last almost 48 hours, thousands of copies of the plasmodium simultaneously reach the same degree of maturity and break out from the red blood cell to invade other healthy cells. Celia, Regina and biochemist, Carlos Hotta, proved that the rate at which the protozoan matures is regulated by a hormone produced by the host’s organism – melatonin, manufactured by a gland at the base of the brain and responsible for adjusting biological rhythms, like sleeping and being awake. Without melatonin, the parasites do not develop at the same time, according to an article published in 2000 in Nature Cell Biology. In 2005, biologist Flavio Beraldo and Celia discovered how melatonin regulates the coordinated development of the parasites: the hormone activates a chain of cell messengers that control the genes linked to multiplication of the plasmodium.
Besides being a curious fact, this orchestrated maturing is arousing clinical interest. In a study to be published in the Open Parasitology Journal, Piero Bagnaresi, from the IBUSP team, shows that it is easier to kill the parasites when they mature in an unsynchronized way: a smaller dose of the medication chloroquinine – whose action mechanism against the parasite was recently explained by Celia and Gazarini – becomes as effective as the drugs normally prescribed in the treatment of malaria.
Analyzing the genome of Plasmodium falciparum, the most lethal form of the protozoan and responsible for 30% of the cases of malaria in Brazil, Celia and Luciana Madeira identified the genes that codify four surface proteins of Plasmodium and which apparently bond with compounds like melatonin, serotonin and other molecules derived from the tryptophane amino-acid, which are essential for the parasite and that can become the target of anti-malarial compounds.
Until new medication appears and no efficient vaccine formula is found the answer is still to eliminate the main transmitter of malaria, which in Brazil is the Anopheles darlingi mosquito, which every year contaminates 500,000 people – almost all of them in the Amazon – with Plasmodium falciparum, the most deadly, or Plasmodium vivax, the most common.
According to specialists the campaigns for fighting Anopheles with engineering works (draining marshes and mangrove swamps) and the use of insecticides were what led to a significant reduction in malaria in Brazil and the world in the last century.
In Brazil the number of infections went down from 4 million a year in the 1930s and 1940s to approximately 50,000 in the 1960s, but started rising in the following decade when the Amazon started being occupied and has stabilized at around 500,000 a year. “There was a reasonable amount of success and outside the Amazon malaria has been practically wiped out, but there’s still a lot to be done”, comments parasitologist, Marcelo Urbano Ferreira, from USP’s Institute of Biomedical Sciences (ICB).
Man and the green
This situation may not improve if nothing is done about the way the land is occupied in the Amazon Forest. Years ago geographer, Márcia Caldas de Castro, a researcher from Princeton University in the United States, evaluated patterns of the spread of malaria in a farming settlement in Machadinho, in Rondônia, and saw that transmission of the disease is very much higher in the early days of the settlement when the forest starts being chopped down. As the land is laid bare the number of cases decreases.
In the article in 2005 in which they describe the results in the Proceedings of the National Academy of Sciences, Castro and her colleagues presented a controversial proposal for reducing the initial transmission outbreak: select only land that is suitable for agriculture for installing settlers and offer them the conditions they need for chopping down the forest as quickly as possible. “In the early phase forest clearance favors transmission, but afterwards it helps prevent it”, explains Ferreira.
Monitoring the transmission dynamic of malaria in another settlement – in the municipality of Acrelândia, in Acre, close to the border with Rondônia -, Ferreira, physician Mônica da Silva Nunes from USP, and Pascoal Muniz, from the Federal University of Acre, also observed that the risk of contracting malaria is higher among people that live in houses close to the forest and work in activities that demand they go into it, as they described in a recent article in the American Journal of Tropical Medicine and Hygiene.
In Acrelândia, Mônica also saw that almost half the cases of malaria were concentrated in just 20% of the houses, a clear sign that control of transmission is possible if appropriate measures are adopted.
What is needed to reduce the number of cases of malaria in Brazil? In Ferreira’s opinion, re-establishing communication between researchers and the people who work in controlling the disease.
“There’s a big gap between the production of knowledge about malaria and application of this knowledge”, says the parasitologist from USP. “The people doing research don’t always produce information that is relevant for prevention and control; and those working in prevention and control often don’t know what the researchers are doing”, says Ferreira, who in October took part in a meeting in which the two groups met. Without dialogue it is probable that malaria will continue to be an obstacle to development, as Dr. Carlos Chagas used to think.
1. Genetic diversity, population structure and the transmission dynamic of Plasmodium vivax in Brazil’s rural Amazon region (nº 07/51199-0); Modality Regular Help with Research; Coordinator Marcelo Urbano Ferreira – ICBUSP; Investment R$ 197,040.00 (FAPESP).
2. Molecular bases of the signal transduction in the cellular cycle of malaria parasites (nº 02/06194-7); Modality Thematic Project; Coordinator Celia Regina Garcia – IBUSP; Investment R$ 1,082,371.51 (FAPESP)
AMINO, R. et al. Quantitative imaging of Plasmodium transmission from mosquito to mammal. Nature Medicine. v. 12. p. 220-224. 2006.
Silva-Nunes, M. et al. Malaria on the Amazonian frontier: transmission, dynamics, risk factors, spatial distribution and prospects for control. American Journal of Tropical Medicine and Hygiene. v. 79. p. 624-635. 2008.