The modification makes Plasmodium falciparum turn fluorescent in the presence of calcium, an ion essential for its survival, and may be useful in the screening of new medicines (photo: Wikimedia)

University of São Paulo scientists create a transgenic version of the malaria parasite
2014-05-21

The modification makes Plasmodium falciparum turn fluorescent in the presence of calcium, an ion essential for its survival, and may be useful in the screening of new medicines.

University of São Paulo scientists create a transgenic version of the malaria parasite

The modification makes Plasmodium falciparum turn fluorescent in the presence of calcium, an ion essential for its survival, and may be useful in the screening of new medicines.

2014-05-21

The modification makes Plasmodium falciparum turn fluorescent in the presence of calcium, an ion essential for its survival, and may be useful in the screening of new medicines (photo: Wikimedia)

 

By Karina Toledo

Agência FAPESP – Researchers at the Biosciences Institute of the University of São Paulo (IB-USP) have developed a transgenic version of the parasite that causes the most aggressive form of malaria affecting humans – Plasmodium falciparum – which may help in screening new medicines to treat the disease.

The findings of the study, which was conducted with funding from FAPESP as part of the doctoral research of Lucas Borges Pereira, were presented on April 15 at the “Workshop at the Interface between Physics and Biology” at the FAPESP headquarters in São Paulo.

Inserted into the parasite was a gene that encodes a calcium marker known as GCaMP3, which is formed by the fusion of green fluorescent protein (GFP, first isolated from a type of jellyfish), the gene for the protein calmodulin (CaM, capable of binding to calcium), and the M13 peptide.

“In the presence of calcium, an interaction occurs that causes the GFP to emit light. The higher the amount of ion, the more fluorescence there is. It is a type of tool used frequently in studies in mammalian cells. The fact that we have managed to develop a version for the parasite that kills the most people in the world is a huge advance,” said Célia Regina da Silva Garcia, an advisor of Borges and coordinator of the thematic project “Functional genomics in Plasmodium.”

Garcia explained that calcium is a substance that P. falciparum must have for reproduction and for the processes involved in invading host cells.

“The great advantage of the transgenic parasite is the possibility of measuring calcium in real time, without using invasive methods such as host cell extraction or organic compound marking. This way, we are able to study the physiological processes involved in controlling the life cycle with the intact cell and live parasite [in vitro]. We’ll also be able to conduct screening for new drugs,” she explained.

In a previous study published in The Journal of Cell Biology, the USP team had discovered how the parasite is able to survive inside erythrocytes – the red cells of the blood – where there is ten thousand times less calcium than what is needed for the parasite’s survival.

At the time that the parasite penetrates an erythrocyte, part of the membrane of the blood cell folds over and forms a sac around the protozoan, called the parasitophorous vacuole. In this way, the parasite creates a calcium-rich environment around itself. One protein in the wall of this sac, the enzyme Ca2+ ATPase, plays an essential role in this process by capturing calcium inside the erythrocyte.

“Drugs like artemisinin, for example, cause the parasite to die by inhibiting its capacity to accumulate calcium. Other antimalarial drugs also act on the calcium signaling channel. However, we need to find new therapeutic options because the parasite is already becoming resistant to the existing drugs,” Garcia said.

The group’s idea is to use the transgenic parasite as a tool in the testing of drug candidates via high-throughput screening (HTS) tests that use an automated system to evaluate several substances at the same time. “Several inhibitors can be placed on the modified parasites and, depending on the intensity of the fluorescence, we can identify which of the substances is more effective,” Garcia explained.

With the help of an instrument known as a flow cytometer, which allows the identification of fluorescent cells, Garcia’s group has already conducted initial tests to confirm the sensitivity of the transgenic parasite to the presence of calcium.

“However, the flow cytometer only allows us to see the population of parasites inside the erythrocytes. In order to study the individual behavior of the Plasmodium, we need more sophisticated equipment, such as a confocal microscope. Only then will we be able to analyze a single cell and to monitor the calcium signaling,” Borges said.

Although partnerships with international teams may be possible, Garcia believes that the next phase of experiments needs to be conducted in Brazil. “These are the tests that will provide high-impact results for our science,” she said.

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