Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites Shailja Singh 1 , M. Mahmood Alam 1 , Ipsita Pal-Bhowmick 2 , Joseph A. Brzostowski 3 , Chetan E. Chitnis 1 * 1 Malaria Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India, 2 Laboratory of Malaria and Vector Research (LMVR), National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America, 3 Laboratory of Immunogenetics Imaging Facility, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Rockville, Maryland, United States of America Abstract The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth. Citation: Singh S, Alam MM, Pal-Bhowmick I, Brzostowski JA, Chitnis CE (2010) Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites. PLoS Pathog 6(2): e1000746. doi:10.1371/journal.ppat.1000746 Editor: Michael John Blackman, National Institute for Medical Research, United Kingdom Received May 11, 2009; Accepted December 31, 2009; Published February 5, 2010 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported in part by core funds from ICGEB, New Delhi, and in part by MalSig project funded by European Commission under FP7. 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: cchitnis@icgeb.res.in Introduction Malaria continues to be a major public health problem in tropical regions of the world. It is responsible for significant morbidity and mortality with around 300 to 500 million malaria cases reported annually that result in about 2 million malaria- related deaths [1]. Of the Plasmodium species responsible for human malaria, Plasmodium falciparum is the most virulent and accounts for the vast majority of deaths attributed to malaria. Given the rapid spread of drug resistant malaria parasites, there is an urgent need to develop novel intervention strategies including new drugs and effective vaccines to combat malaria. All the clinical symptoms of malaria are attributed to the blood stage of the parasite life cycle during which Plasmodium merozoites invade and multiply within host erythrocytes. Invasion of erythrocytes by Plasmodium merozoites is a complex multi-step process that is mediated by specific molecular interactions between host receptors and parasite ligands [2]. A clear understanding of the molecular mechanisms involved in erythrocyte invasion could lead to the development of novel approaches to inhibit invasion, limit blood-stage parasite growth and protect against malaria. Plasmodium species belong to the phylum Apicomplexa and are characterized by the presence of apical membrane bound organelles called micronemes and rhoptries that play important roles in host cell invasion. A number of key parasite ligands that mediate critical interactions with host receptors during invasion are localized in these apical organelles [2]. Studies of the invasion process by light and electron microscopy suggest that the early steps of invasion include merozoite attachment to the erythrocyte surface, apical re-orientation, release of apical organelles including micronemes and rhoptries and formation of an irreversible ‘junction’ between the apical end of the invading merozoite and the target erythrocyte [3–6]. The precise timing and sequence of microneme and rhoptry secretion during invasion is not known. Moreover, the external signals and signaling pathways that trigger the coordinated release of microneme and rhoptry proteins remain to be identified. In case of Plasmodium sporozoites cAMP as well as cytosolic Ca +2 have been shown to be involved in signaling mechanisms PLoS Pathogens | www.plospathogens.org 1 February 2010 | Volume 6 | Issue 2 | e1000746