2+ Blockage of Spontaneous Ca Oscillation Causes Cell Death in Intraerythrocitic Plasmodium falciparum Masahiro Enomoto1.¤, Shin-ichiro Kawazu2., Satoru Kawai3, Wakako Furuyama 2 , Tohru Ikegami4, Jun- ichi Watanabe4{, Katsuhiko Mikoshiba1,5* 1 Laboratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako-shi, Saitama, Japan, 2 National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan, 3 Laboratory of Tropical Medicine and Parasitology, Dokkyo Medical University, Mibu, Tochigi, Japan, 4 Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan, 5 Japan Science and Technology Agency, International Cooperative Research Project and Solution-Oriented Research for Science and Technology, Calcium Oscillation Project, Saitama, Japan Abstract Malaria remains one of the world’s most important infectious diseases and is responsible for enormous mortality and morbidity. Resistance to antimalarial drugs is a challenging problem in malaria control. Clinical malaria is associated with the proliferation and development of Plasmodium parasites in human erythrocytes. Especially, the development into the mature forms (trophozoite and schizont) of Plasmodium falciparum (P. falciparum) causes severe malaria symptoms due to a distinctive property, sequestration which is not shared by any other human malaria. Ca2+ is well known to be a highly versatile intracellular messenger that regulates many different cellular processes. Cytosolic Ca2+ increases evoked by extracellular stimuli are often observed in the form of oscillating Ca2+ spikes (Ca2+ oscillation) in eukaryotic cells. However, in lower eukaryotic and plant cells the physiological roles and the molecular mechanisms of Ca2+ oscillation are poorly understood. Here, we showed the observation of the inositol 1,4,5-trisphospate (IP3)-dependent spontaneous Ca2+ oscillation in P. falciparum without any exogenous extracellular stimulation by using live cell fluorescence Ca2+ imaging. Intraerythrocytic P. falciparum exhibited stage-specific Ca2+ oscillations in ring form and trophozoite stages which were blocked by IP3 receptor inhibitor, 2-aminoethyl diphenylborinate (2-APB). Analyses of parasitaemia and parasite size and electron micrograph of 2-APB-treated P. falciparum revealed that 2-APB severely obstructed the intraerythrocytic maturation, resulting in cell death of the parasites. Furthermore, we confirmed the similar lethal effect of 2-APB on the chloroquine-resistant strain of P. falciparum. To our best knowledge, we for the first time showed the existence of the spontaneous Ca2+ oscillation in Plasmodium species and clearly demonstrated that IP3-dependent spontaneous Ca2+ oscillation in P. falciparum is critical for the development of the blood stage of the parasites. Our results provide a novel concept that IP3/Ca2+ signaling pathway in the intraerythrocytic malaria parasites is a promising target for antimalarial drug development. Citation: Enomoto M, Kawazu S-i, Kawai S, Furuyama W, Ikegami T, et al. (2012) Blockage of Spontaneous Ca2+ Oscillation Causes Cell Death in Intraerythrocitic Plasmodium falciparum. PLoS ONE 7(7): e39499. doi:10.1371/journal.pone.0039499 Editor: Georges Snounou, Universite´ Pierre et Marie Curie, France Received January 15, 2012; Accepted May 21, 2012; Published July 6, 2012 Copyright: 2012 Enomoto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to K.M. from the Japan Science and Technology Agency and the Ministry of Education, Culture, Sports, Science and Technology of Japan (2022007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mikosiba@brain.riken.jp . These authors contributed equally to this work. ¤ Current address: Division of Signaling Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada { Deceased Introduction Malaria continues to be a worldwide public health problem causing significant morbidity and mortality and its resistance to existing antimalarial drugs is a growing problem [1]. The life cycle of Plasmodium species is complex (Fig. 1). Infection of humans begins with a small inoculum of sporozoites from the salivary glands of a blood-feeding Anopheles mosquito. Sporozoites pene- trate liver cells, transform and multiply asexually to produce thousands of free merozoites (liver stage). Each of these asexual merozoites invades an erythrocyte and enters into another phase of asexual reproduction, and then bursts the cell, releasing 8–32 more merozoites to invade more erythrocytes (blood stage). In infected erythrocytes, development of the parasites is accompanied by morphological changes such as ring form, trophozoite and schizont stages. P falciparum is responsible for the lethal form of human malaria. The mature forms of the intraerythrocytic parasite (trophozoite and schizont) remodel the cytoskeleton and plasma membrane to create cytoadherence knobs as well as nutrient permeation pathways and alter the mechanical stability of the erythrocytes, causing them to stick to blood vessels [2,3]. This leads to blockage of the microcirculation and results in dysfunction of multiple organs, typically the brain in cerebral malaria [4]. Calcium (Ca2+) oscillations are ubiquitous intracellular signals responsible for numerous cellular processes, including excitation– contraction coupling, fertilization, cell growth, transformation, secretion and neuronal signalling [5,6]. In apicomplexan parasites, such as Plasmodium, Toxoplasma and Cryptosporidium, Ca2+-mediated PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e39499