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Calcium role in FIS1 and DRP1 genes activation on mitochondrial division of P. falciparum (WT) and parasite knockout strains for kinase PfPK7

Grant number: 16/09185-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): June 01, 2016
Effective date (End): October 31, 2017
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Célia Regina da Silva Garcia
Grantee:Camila Lie Kiyan
Home 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 an endemic disease in many tropical countries and it's caused by a parasite from apicomplexa phylum (Plasmodium) and transmitted by the bite of Anopheles mosquitoes. In humans, the most severe form of the disease is caused by Plasmodium falciparum. The disease kills 2 to 3 million people annually; mortality gets to be so high that it is considered the largest among the parasitic diseases (Snow, Guerra et al. 2005). It's necessary obtaining new drugs once it has already been observed parasites resistant to all known antimalarial drugs, so research for a better understanding of parasite biology is made necessary for the development of effective drugs and vaccines to combat the disease (Mita and Tanabe, 2012). The parasite's life cycle occurs in two hosts: one vertebrate and other invertebrate. Thus, Plasmodium is a parasite that is able to perceive the host´s microenvironment and have mechanisms that enable using intracellular signaling pathway to modulate several processes. Calcium is one of these flags and is able to act in processes ranging from exocytosis to cell proliferation by increasing or decreasing their intracellular concentration. However its homeostasis must be maintained, because high concentrations of calcium maintained for a long time cause cell apoptosis (Berridge, 2001; Berridge et al., 2003). The falciparum strain obtained by knockout of the kinase protein 7 gene (PfPK7) is unable to perceive the microenvironment like wild parasites (3D7 strain) do, not responding to melatonin stimuli and therefore unable to obtain a synchronous cycle (Koyama et al, 2012). Therefore this strain becomes a great tool for studying signal transduction mechanisms that are responsible for changing the progression of the parasite cycle. It is known that the mitochondria of the parasite is capable of reversibly accumulate part of the calcium from the cytoplasm, participating in homeostasis maintenance of this ion in the parasite and ensuring its survival (Gazarini; Garcia 2004). The P. falciparum mitochondria have unusual evolutionary and functional characteristics, since the electron transport chain (ETC) is not its main source of ATP. So far it was only identified three proteins of CTE encoded by the mitochondrial genome, and some ribosomal RNA fragments (Vaidya, Mather 2009). Nevertheless, CTE inhibitors, such as atovaquone antimalarial drug, proved lethal to the parasite development and therefore leads to the conclusion that the mitochondria should participate in vital physiological processes in Plasmodium falciparum (Painter et al . 2007). In this context, we have shown the importance of studying the role of mitochondria in the division and cell signaling mechanisms of Plasmodium falciparum during schizogony. The objective of this project is to analyze the expression of two major genes related to mitochondrial fission (FIS1 and Drp1) in strains of Plasmodium falciparum 3D7 and PfPK7 during intraeritrocítico cycle in the presence and absence of Ionomicin, an ionophore for Ca2+. Furthermore, the number and morphology of mitochondria will be examined and related to the expression of these proteins in these two strains, using chemiluminescence and microscopy techniques at all stages of parasite development in the presence and absence of Ca2+ stimulus. (AU)