Dissect calcium signaling pathways in Plasmodium falciparum using parasites that express the genetic marker for calcium, PfGCaMP3

Malaria is a disease caused by the parasite of the genus Plasmodium, being responsible for more than 1 million deaths per year (Snow et al., 2005). The species P. falciparum is responsible for the most severe form of the disease (Miller et al., 2002). The life cycle of Plasmodium alternates between two hosts: (I) Anopheles mosquito, where sexual reproduction occurs, with the formation of sporozoites (II) and vertebrate, where Plasmodium invades hepatocytes and erythrocytes, developing in the stages ring, trophozoite and schizont. With the breakdown of erythrocytes, the merozoites, released into the bloodstream, invade other erythrocytes, restarting the cycle. The signaling mechanism involved in the process of differentiating merozoites into gametocytes, a form that infects the mosquito, during the insect’s blood meal phase is not known. During the life cycle, the parasite needs to perceive and adapt to different ionic environments, which include the intra and extracellular environment in very phylogenetically distant hosts (Bannister and Mitchell, 2003). From the work of several groups, and the sequencing of the Plasmodium genome, it is well accepted that the parasite expresses several genes that encode the expression of proteins involved in signal transduction machinery. However, it is not known how this signaling modulates cellular processes, such as cell cycle progression, red cell invasion, differentiation into gametocytes and egress of the host cell. Ca2 + is an ubiquitous intracellular signaler that regulates several cellular functions (Berridge et al., 2003). As in any eukaryotic cell, the malaria parasite maintains a low cytoplasmic concentration of Ca2 + (Budu and Garcia, 2012, Lew and Tiffert, 2007). Studies of signal transduction mechanisms have identified melatonin, UTP, ATP, among others, as signaling molecules relevant for controlling the cell division of Plasmodium falciparum (Hotta and at, 2000, Koyama et al, 2009). At what stage of the Plasmodium cycle, these molecules come into play and how they regulate the cell cycle, it is not completely known. Mammalian cells that express genetically encoded calcium indicators have been widely used to study cell physiology in different processes. Recently, our group constructed the transgenic parasite PfGCaMP3 (Borges-Pereira et al., 2014), capable of monitoring fluctuations in calcium concentration, without requiring invasive methods, such as the introduction of chemical fluorescent markers. In this way, PfGCaMP3 is an excellent tool that will allow monitoring cytosolic molecular events in Plasmodium falciparum that involve Ca2 + as a signal. This work aims to study in detail the signaling pathways involving Ca2 + in P. falciparum, using the transgenic parasite PfGCaMP3 to study the dynamics of this ion. One of the advantages for using PfGCaMP3 is that it will not be necessary to remove the red blood cell, since GCaMP3 is expressed only in Plasmodium. The parasite expressing GCaMP3 will be used to measure calcium signaling mechanisms in the parasite development stages – ring, trophozoite and schizont. For this, calcium ionophore (ionomycin) and potassium (nigericin) will be used. Drugs will also be used to dissect signaling pathways in the parasite in a physiological state, inside the red blood cell, to understand the role of mitochondria, endoplasmic reticulum and acidocalcisoma in maintaining calcium homeostasis during the asexual development of Plasmodium.

Project details

Basic Science
Genetics and Genomics

Jun 2016 — May 2017

Brazil