Cysteine-protease activity elicited by Ca²⁺ stimulus in Plasmodium

Abstract

Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653–3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca²⁺ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca²⁺ chelator, BAPTA. When parasites were treated with cytoplasmic Ca²⁺ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH₂, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca²⁺.

Introduction

Malaria is one of the most important infectious diseases in the world, being responsible for more than one million deaths each year, most of them children under five (WHO, www.rbm.who.int). The cell cycle of Plasmodium within red blood cells (RBCs) is marked by its 24 h multiplicity [1], [2] according to the host circadian rhythm. The host melatonin hormone peak during the night period [3], [4] has been shown to modulate the Plasmodium cell cycle through a cytoplasmic Ca²⁺ increase resulting in parasite synchronization [5], [6]. Calcium homeostatic control permits a series of related processes to be readily turned on and off in response to ion mobilization from the major intracellular calcium reservoirs, the endoplasmic reticulum, mitochondria and acidic compartments [7], [8], [9]. Molecular and cellular studies on Ca²⁺ signaling in Plasmodium parasites [10], [11] have revealed the Ca²⁺ storage organelles to include the endoplasmic reticulum based in its sensitivity to Ca²⁺-ATPase inhibitors such as thapsigargin and ciclopiazonic acid [12], [13], [14], [15], [16], [17], [18], [19] and acidic Ca²⁺ pool [15], [16], [17], [20]. In addition, mitochondria are thought to play a role in Ca²⁺ homeostatic control in these parasites [21]. Exposure to a high extracellular Ca²⁺ concentration might create appropriate conditions for the use of Ca²⁺ as a second messenger in the modulation of parasite intracellular signaling events [22].

Downstream mechanisms related to melatonin and Ca²⁺ signaling might involve molecular changes such as protein activation or switches in gene expression in the parasites. Such events usually involve remodeling proteolysis [23], [24], [25], [26], a key-driving element able to activate or inactivate intermediate regulating factors, as it occurs with cyclins in the progression of cell cycle in mammals [27], [28].

Cysteine-proteases are involved in the progression of the intraerythrocytic life cycle, with roles in degradation of hemoglobin [26], [29], [30], [31] and erythrocyte cytoskeletal proteins, such as ankyrin and band 4.1 [32], and erythrocyte rupture [33]. We report in this work the presence of a cysteine-protease activity elicited by Ca²⁺-in malaria parasites, which was detected by hydrolysis of FRET peptides and inhibited by an irreversible fluorescent-labeled cysteine-protease inhibitor. The FRET peptides and the inhibitor Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH₂ (Abz-peptidyl-epoxide) were incorporated by the parasites and the hydrolytic activity or enzyme labeling by inhibitor were observed at the same time as the cytoplasmic Ca²⁺ release. The loading of peptides into free parasites and parasites inside RBC have been described previously [34]. Here we further investigate the permeation of peptides into the Plasmodium developmental stages (ring, trophozoite, and schizont) using fluorescent Abz- and FITC-peptides of molecular weight ranging from 653 to 3146 Da.