Abstract
A model betalainic dye was semisynthesized from betanin, the magenta pigment of the red beet, and was effective for live-cell imaging of Plasmodium-infected red blood cells. This water-soluble fluorescent probe is photostable, excitable in the visible region and cell membrane-permeable, and its photophysical properties are not notably pH-sensitive. Fluorescence imaging microscopy of erythrocytes infected with Plasmodium falciparum, a causative agent of malaria in humans, showed that only the parasite was stained. Z-stacking analysis suggested that the probe accumulates proximal to the nucleus of the parasite. Indicaxanthin, one of the natural fluorescent betalains found in the petals of certain flowers, did not stain the parasite or the red blood cell.
Highlights
[1] The biosynthesis of betalains is based on the oxidative cleavage of 3,4-dihydroxyphenylalanine (DOPA) in the presence of DOPA 4,5-dioxygenase, which is encoded by the BvDODA1 gene [2], followed by a spontaneous aldimine coupling between the resulting betalamic acid (HBt) and amines or amino acids
Differentiation inside the erythrocyte occurs within the parasitophorous vacuole (PV), a vacuolar membrane that surrounds the intracellular parasite
The sequential pathway for the entry of exogenous nutrients required for parasite survival and multiplication includes crossing the red blood cell membrane (RBCM), the parasitophorous vacuole membrane and the parasite plasma membrane
Summary
Betalains are water-soluble pigments responsible for the visible fluorescence of flowering succulent plants. [1] The biosynthesis of betalains is based on the oxidative cleavage of 3,4-dihydroxyphenylalanine (DOPA) in the presence of DOPA 4,5-dioxygenase, which is encoded by the BvDODA1 gene [2], followed by a spontaneous aldimine coupling between the resulting betalamic acid (HBt) and amines or amino acids. [3] Natural betalains are non-toxic antioxidants known to bind to biological membranes and interact with human lipoproteins, most likely due to their conjugated amphiphilic chemical structures (Scheme S1). [4] Extracts of plants pigmented with betalains have been used in popular medicine for the treatment of a variety of diseases including malaria, [5] a severe infectious disease responsible for millions of deaths each year worldwide. [6]. Plasmodium species are the causative agents of malaria These obligate, intracellular organisms have a complex life cycle and switch between a mosquito vector and a vertebrate host. [7] Plasmodium spp. merozoites infect erythrocytes and undergo a differentiation process that starts with the ring stage, followed by the trophozoite stage and the schizont stage. Differentiation inside the erythrocyte occurs within the parasitophorous vacuole (PV), a vacuolar membrane that surrounds the intracellular parasite. Inspired by the biological role of betalains, we sought to create a new small, tunable and water-soluble molecular framework for dyes suitable for live-cell fluorescence imaging. Due to the presence of at least two carboxyl groups in the betalain framework, such dyes are negatively charged at near-neutral pH and favor cell permeation through anion channels, such as the NPP. This dye was used for the identification of erythrocytes infected by Plasmodium spp. using fluorescence imaging
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