Methods Used to Investigate the Plasmodium falciparum Digestive Vacuole

Abstract

Plasmodium falciparum malaria remains a global health problem as parasites continue to develop resistance to all antimalarials in use. Infection causes clinical symptoms during the intra-erythrocytic stage of the lifecycle where the parasite infects and replicates within red blood cells (RBC). During this stage, P. falciparum digests the main constituent of the RBC, hemoglobin, in a specialized acidic compartment termed the digestive vacuole (DV), a process essential for survival. Many therapeutics in use target one or multiple aspects of the DV, with chloroquine and its derivatives, as well as artemisinin, having mechanisms of action within this organelle. In order to better understand how current therapeutics and those under development target DV processes, techniques used to investigate the DV are paramount. This review outlines the involvement of the DV in therapeutics currently in use and focuses on the range of techniques that are currently utilized to study this organelle including microscopy, biochemical analysis, genetic approaches and metabolomic studies. Importantly, continued development and application of these techniques will aid in our understanding of the DV and in the development of new therapeutics or therapeutic partners for the future.

Keywords: Plasmodium falciparum; antimalarial; digestive vacuole; hemoglobin digestion; microscopy.

Figure 1

Overview of Plasmodium falciparum lifecycle, the digestive vacuole (DV) and the effect of antimalarials. (A) Upon injection into the human host, P. falciparum parasites initially infect and then go through a developmental phase in the liver. Parasites are then released into the blood as merozoites where they undergo the intra-erythrocytic cycle. Here, they invade red blood cells (RBC), then develop into ring stage parasites, followed by trophozoite and finally schizont stages where new merozoites are formed. Upon rupturing of the schizont, merozoites are released to propagate the cycle. (B) By the trophozoite stage, the digestive vacuole is visible. RBC hemoglobin (Florens et al., 2002) is taken up by the parasite through a cytostome (pink tear-shaped) and delivered to the digestive vacuole (grey) where it undergoes digestion by a number of enzymes (see key; pac-man). Small peptides are transported through the chloroquine resistant transporter (CRT) and are further digested into free amino acids. Heme (grey globin chains) is toxic and is crystallized into hemozoin (Hz) through unknown mechanisms. Chloroquine (CQ) acts by blocking the formation of Hz causing parasite death but can be overcome by mutant forms of CRT (CRT (R)) that are able to transport CQ out of the digestive vacuole. Artemisinin (Art) is activated by heme (Art*) which causes free radical damage to many organelles including the nucleus (N) and mitochondria (M). Resistance to Art is mediated by Kelch-13 (K-13), the latter involved in Hb endocytosis and trafficking to the DV. A, apicoplast; PVM, parasitophorous vacuole membrane; PPM, parasite plasma membrane.