Malaria parasites, Plasmodia, spend most of their asexual life cycle within red blood cells, where they proliferate and mature. The erythrocyte cytoplasm has very low [Ca²⁺] (<100 nM), which is very different from the extracellular environment encountered by most eukaryotic cells. The absence of extracellular Ca²⁺ is usually incompatible with normal cell functions and survival. In the present work, we have tested the possibility that Plasmodia overcome the limitation posed by the erythrocyte intracellular environment through the maintenance of a high [Ca²⁺] within the parasitophorous vacuole (PV), the compartment formed during invasion and within which the parasites grow and divide. Thus, Plasmodia were allowed to invade erythrocytes in the presence of Ca²⁺ indicator dyes. This allowed selective loading of the Ca²⁺ probes within the PV. The [Ca²⁺] within this compartment was found to be ∼40 μM, i.e., high enough to be compatible with a normal loading of the Plasmodia intracellular Ca²⁺ stores, a prerequisite for the use of a Ca²⁺-based signaling mechanism. We also show that reduction of extracellular [Ca²⁺] results in a slow depletion of the [Ca²⁺] within the PV. A transient drop of [Ca²⁺] in the PV for a period as short as 2 h affects the maturation process of the parasites within the erythrocytes, with a major reduction 48 h later in the percentage of schizonts, the form that re-invades the red blood cells.

Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. All carotenoids are derived from the C40 isoprenoid precursor geranylgeranyl pyrophosphate, and their chemical and physical properties are associated with light absorption, free radical scavenging, and antioxidant activity. Carotenoids are generally synthesized in well defined subcellular organelles, the plastids, which are also present in the phylum Apicomplexa, which comprises a number of important human parasites, such as Plasmodium and Toxoplasma. Recently, it was demonstrated that Toxoplasma gondii synthesizes abscisic acid. We therefore asked if Plasmodium falciparum is also capable of synthesizing carotenoids. Herein, biochemical findings demonstrated the presence of carotenoid biosynthesis in the intraerythrocytic stages of the apicomplexan parasite P. falciparum. Using metabolic labeling with radioisotopes, in vitro inhibition tests with norflurazon, a specific inhibitor of plant carotenoid biosynthesis, the results showed that intraerythrocytic stages of P. falciparum synthesize carotenoid compounds. A plasmodial enzyme that presented phytoene synthase activity was also identified and characterized. These findings not only contribute to the current understanding of P. falciparum evolution but shed light on a pathway that could serve as a chemotherapeutic target.