Plasmodium falciparum, which causes the most aggressive form of malaria, is a versatile parasite. In the host organism, the parasite lodges first in skin and liver cells, where it matures and multiplies, before entering the bloodstream and invading the red blood cells (erythrocytes). It is within the erythrocytes, however, that the parasite performs feats that allow it to stay alive and get rid of toxic waste it produces as it feeds on the host. In an article published in late February 2016 in the journal Scientific Reports, British and Brazilian researchers, coordinated by biochemist Célia Garcia, of the Biosciences Institute of the University of São Paulo (IB-USP), described a new biochemical strategy the parasite uses to eliminate such waste and thus survive and mature within the erythrocytes. According to the researchers, the mechanism they identified, combined with another that has been known for some time, can enhance the prospects of developing new strategies to combat malaria.
For at least two decades Garcia’s team has been investigating what happens to Plasmodium after it installs itself in red blood cells. During this period, she and her team found that one of the secrets that allows the parasite to survive within the erythrocytes is related to the way in which it invades them. Instead of piercing the membrane, Plasmodium simply pushes against it. Because of the membrane’s elasticity, it deforms and surrounds Plasmodium, forming a pocket around it where the concentration of calcium is higher than inside the cell and mimics blood plasma—calcium is an important element for the survival of the parasite. Plasmodium then multiplies and passes through three stages of development. After 48 hours, each of the thousands of copies of the parasite reach the same degree of maturity and disrupt the red blood cells, going on to invade healthy erythrocytes. In previous studies, the biochemical group had also found that the parasite maturation rate is regulated by a hormone produced by the host organism, melatonin, which in mammals controls the cycles of sleep and wakefulness (see Pesquisa FAPESP Issue nº 153).
The parasite survives inside red blood cells by feeding on hemoglobin, the protein responsible for transporting oxygen and giving blood its red color. It has been known for some time that to do this the parasite produces an enzyme that breaks this molecule into smaller pieces, the amino acids. Garcia explains that a molecule called heme results from this process, which, if not removed, can become toxic and damage the cells and the very parasite that produced it.
In the 1980s, researchers found that as it evolved, P. falciparum developed at least one way of protecting itself from this toxic substance, turning it into a harmless polymer, hemozoin. This mechanism is now the main target of chloroquine, the most widely used antimalarial drug in the world. By preventing the formation of this polymer, chloroquine inhibits the growth and reproduction of the parasite inside the erythrocytes. The problem is that in recent decades the drug has become less effective against Plasmodium, particularly in South America and Southeast Asia.
In 2010, Garcia’s group observed that another mechanism—common in mammalian bodies, but previously unknown in Plasmodium—also allows the parasite to neutralize the heme group. In a study published in the journal Cell Biology International, the USP researchers found that the parasite produces an enzyme called heme-oxygenase, which converts heme into biliverdin, a molecule that is non-toxic at low concentrations. At certain levels, however, biliverdin can become harmful to the parasite. “Converting the heme into biliverdin, instead of turning it into a polymer, may pose a risk to the survival of the parasite within the red blood cells,” says Garcia.
Researchers do not know under what circumstances determine which of the two ways Plasmodium will use to neutralize the toxic compounds. One hypothesis, according to Garcia, is that the second strategy would slow the parasite’s life cycle, reducing its metabolism and the production of these harmful substances.
In the study now published in Scientific Reports, researchers investigated the role of biliverdin in the life cycle of the parasite, which infects 250 million people worldwide each year (and kills almost one million of them, mostly children), especially in Africa, Asia and Latin America. In collaboration with Rita Tewari, of the University of Nottingham in England, Garcia’s group interrupted the gene expression involved in the production of heme-oxygenase in parasites of the species P. berghei, preventing them from producing biliverdin. Although not the main route used by the parasite to neutralize toxic substances, its production appears to be important for the life of the parasite. “When we suppressed the gene expression that produces heme oxygenase, the parasite died,” says Garcia, who was surprised by the result. We showed that P. berghei, which infects rodents, is unable to develop within erythrocytes if it can not produce heme-oxygenase,” she says. We still have to see if the same happens in the case of P. falciparum. Garcia thinks this may be a way to create new strategies to combat the parasite.
Apparently, biliverdin prevents the parasite from maturing in erythrocytes. In the study, researchers conducted a series of laboratory tests in which they observed that biliverdin binds to enolase, an enzyme used by the parasite to produce energy. The discovery surprised them because enolase is part of another Plasmodium biochemical pathway and because, in principle, it should not bind to biliverdin. Garcia and her collaborators, including Glaucius Oliva and Rafael Guido, both of USP in São Carlos, concluded that by binding to enolase, biliverdin reduces Plasmodium’s multiplication rate. “Biliverdin acts as a communications molecule, and enolase as a sensor that detects biliverdin,” suggests Garcia.
Functional genomics in Plasmodium (nº 2011/51295-5); Grant Mechanism Thematic Research Grant; Principal Investigator Célia Regina da Silva Garcia (IB-USP); Investment R$2,068,020.
ALVES, E. et al. Biliverdin targets enolase and eukaryotic initiation factor 2 (eIF2α) to reduce the growth of intraerythrocytic development of the malaria parasite Plasmodium falciparum. Scientific Reports. V. 6, No. 22093, pp. 1-12. February 2016.
SARTORELLO, R. et al. In vivo uptake of a heme analogue Zn protoporphyrin IX by the human malaria parasite P. falciparum-infected red blood cells. Cell Biology International. V. 34, No. 8. pp. 859-65. August 2010.