Inhibition of malaria parasite Plasmodium falciparum development by crotamine, a cell penetrating peptide from the snake venom

Highlights

• Crotamine is a cationic natural peptide with anti-plasmodial activity.

• Crotamine has anti-plasmodial activity against Plasmodium falciparum.

• Crotamine inhibits the development of the P. falciparum in a dose-dependent manner.

• Crotamine was observed in the parasite nucleus and parasitophorous vacuole.

• Crotamine may disrupt the parasite acidic compartments H⁺ homeostasis.

Abstract

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC₅₀ value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H⁺ homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Introduction

Malaria is a critical human infection and it is responsible for the death of nearly a million people every year [1]. The search for new antimalarials compounds is crucial, since drug resistance is spreading quickly in the existing parasite population [2], [3], [4], [5]. In contrast to the increased number of methods currently available to identify new compounds with antimalarial activity as described in recent reports, actually very few innovative contributions to drug discovery can be found in the field [6].

The Plasmodium life cycle involves two well-known hosts, the arthropod mosquito vector and the vertebrate host, for the sexual and asexual stages, respectively. During the asexual stage, which is the main target for the antimalarial studies, crucial biochemical and physiological changes, necessary for the P. falciparum development, are observed in erythrocytes [7], [8], [9]. The increased permeability of the erythrocyte membrane to different metabolites during the asexual stage favors the entrance of several inhibitors with potential as antimalarial drugs [10], [11], [12], [13], [14], [15].

The acidic compartments, in addition to the endoplasmic reticulum and mitochondria, present in Plasmodium cells, possess an important role in the intracellular ionic homeostasis (i.e. Ca²⁺ and H⁺) [9], [16], [17]. They are also the key elements to provide, during the Plasmodium development, the necessary environment for the functions of vital enzymes [18]. For instance, the hemoglobin degradation, which is dependent of the action of different proteases as falcipains and plasmepsinas, occurs in acidic compartments, and therefore, the ion homeostasis is undoubtedly crucial [8], [19], [20]. The acidic compartments are also described as the local of accumulation of antimalarials, as chloroquine and derivatives, which kill the parasite by altering the hemoglobin metabolism and the formation of hemozoin crystals [21]. However, the increasing widespread resistance to chloroquine claims the search for alternative compounds able to inhibit the parasite development [22].

Crotamine is a polypeptide of 42 amino acid residues (sequence YKQCHKKGGHCFPKEKICLPPSSDFGKMDCRWRWKCCKKGSG, MW = 4889.81 Da and pI of about 9.5) isolated from the venom of the South American rattlesnake Crotalus durissus terrificus [23]. The high content of basic amino acid residues and the presence of three disulfide bonds confer to this toxin a stable 3D structure with high exposure of positive charges and an amphipathic characteristic [24], [25]. These structural features stimulated us to investigate the cell penetrating properties of crotamine, which showed a unique high specificity and affinity for actively proliferating cells compared to quiescent non-proliferating or non-tumoral mammalian cells [26], [27], [28], [29]. In addition, crotamine is also capable of carrying genes and other molecules into cells [30], [31], [32]. Taking this into account, the commercial use of crotamine as a biomarker of proliferating cells and/or its employment as a carrier of bioactive molecules into cells was already protected by our group (PCT/BR06/000052; US-2008-0181849-A1; 1866332 EPO Bulletin No. 37/14 in Sept 10th, 2014).

Biochemical, molecular and cellular studies showed that the cell penetrating ability of crotamine is dependent on its positive net charge and its affinity for negatively charged surfaces [30], [31]. Moreover, crotamine also shows cytotoxic effect that involves the disruption of lysosomes and the consequent release of the vesicles contents, as the free calcium and cathepsins, which may trigger cell apoptosis and leading us to suggest the acid compartments of cells as the primary intracellular target of crotamine [31], [33]. Crotamine also presents specificity towards the cell membranes of microorganisms, which is also dependent on their membrane surface negative charges [27], [34], reinforcing again the importance of the negative net charges on surface for the biological activities and functions of crotamine [34].

Therefore, considering that Plasmodium acidic compartments are the local of accumulation of antimalarials and that their ionic homeostasis are crucial for the parasite development, in the present study, the potential antimalarial effect of crotamine was explored in a P. falciparum model. Both selective internalization into infected red blood cells (iRBCs) and intracellular localization of crotamine were visualized by confocal microscopy. In addition, crotamine affected parasite development in a dose-dependent manner, more likely due to the disruption of the P. falciparum parasites H⁺ homeostasis, as evaluated by flow cytometry and by the observed changes in lysosomotropic acridine orange (AO) fluorescence in infected erythrocytes, respectively. Therefore, we believe that the results presented here provide interesting insight for a novel potential structural model for the development of new antimalarials peptidomimetics based on disruption of the H⁺ homeostasis of P. falciparum parasites.