Calcium signaling pathways dissection in Plasmodium falciparum by Pf GCaMP3 calcium-genetic marker parasites

• Grant number: 16/08921-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: June 01, 2016
• End date: May 31, 2017
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Célia Regina da Silva Garcia
• Grantee: Ricardo Mendes Cresta
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 11/51295-5 - Functional genomics in Plasmodium, AP.TEM

Abstract

Malaria is a disease caused by parasites of the genus Plasmodium, accounting 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) mosquito Anopheles, where there is sexual reproduction, with the formation of sporozoites (II) and vertebrate, where the Plasmodium invades hepatocytes and erythrocytes, developing into the stages ring, trophozoite and schizont. With the breakdown of red blood cells, the merozoites, released into the bloodstream, invade other red blood cells, restarting the cycle. It is unknown which signaling mechanism is involved the differentiation process that turns merozoites into gametocytes, which infect the mosquito, during the phase of insect blood meal. During its life cycle, the parasite needs to adapt to different ionic environments that include intra and extracellular medium of phylogenetically very distant hosts (Bannister and Mitchell, 2003). From the research of several working groups, and Plasmodium genome sequencing, it is well accepted that the parasite expresses many genes encoding the expression of proteins involved in signal transduction machinery. However, it is not known how this signal modulates cellular processes such as cell cycle progression, erythrocyte invasion, differentiation into gametocytes and egress from the host cell.Ca2 + is a ubiquitous intracellular signaling that regulates diverse cellular functions (Berridge et al., 2003). Like any other eukaryotic cell, the malaria parasite maintains a low cytoplasmic Ca2 + concentration (Budu and Garcia, 2012, Lew and Tiffert, 2007). Studies on signal transduction mechanisms identified melatonin, UTP, ATP, among others, as relevant signaling molecules to control cell division on Plasmodium falciparum (Hotta and at 2000, Koyama et al, 2009). At which stage of the Plasmodium cycle these molecules come into play and how they regulate the cell cycle, it is not completely known. Mammalian cells expressing genetically encoded calcium indicators have been widely used to study cellular physiology in different processes. Recently, our group built the transgenic parasite PfGCaMP3 (Borges-Pereira et al., 2014), able to monitor fluctuations in the concentration of calcium, without requiring invasive methods, such as introduction of chemical fluorescent markers. Thus the PfGCaMP3 is an excellent tool that will be used to monitor cytosolic molecular events in Plasmodium falciparum involving Ca2 + as flag. This work aims at detailed study of signaling pathways involving Ca2 + in P. falciparum using transgenic parasite PfGCaMP3 to study the dynamics of this ion. One of the advantages to using PfGCaMP3 is that it is not necessary to remove the red blood cell, since the GCaMP3 is expressed only in Plasmodium. The parasite expressing GCaMP3 will be used to measure calcium signaling mechanisms in the parasite's development stages - ring, trophozoite and schizont. For this, it will be used calcium ionophore (ionomycin) and potassium (nigericin). There will also be used drugs to dissect the signalling pathways of the parasite in its physiological state, inside the red blood cell, to understand the role of mitochondria, endoplasmic reticulum and acidocalcissoma in maintaining calcium homeostasis in the assexual development stage of Plasmodium. (AU)