A Nature-Inspired Betalainic Probe for Live-Cell Imaging of Plasmodium-Infected Erythrocytes

PLOS ONE, Dec 2019

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.

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A Nature-Inspired Betalainic Probe for Live-Cell Imaging of Plasmodium-Infected Erythrocytes

et al. (2013) A Nature-Inspired Betalainic Probe for Live-Cell Imaging of Plasmodium- Infected Erythrocytes. PLoS ONE 8(1): e53874. doi:10.1371/journal.pone.0053874 A Nature-Inspired Betalainic Probe for Live-Cell Imaging of Plasmodium -Infected Erythrocytes Letcia Christina Pires Gonc alves 0 Renata Rosito Tonelli 0 Piero Bagnaresi 0 Renato Arruda Mortara 0 Antonio Gilberto Ferreira 0 Erick Leite Bastos 0 Markus Sauer, Julius-Maximilians-University Wu rzburg, Germany 0 1 Centro de Ciencias Naturais e Humanas, Universidade Federal do ABC , Santo Andre , Sao Paulo , Brazil , 2 Departamento de Ciencias Biol o gicas, Universidade Federal de Sa o Paulo , Diadema, Sao Paulo , Brazil , 3 Departamento de Biof sica, Universidade Federal de Sa o Paulo , Sa o Paulo , Brazil , 4 Departamento de Microbiologia, Imunobiologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de Sa o Paulo , Sa o Paulo , Brazil , 5 Departamento de Qu mica, Universidade Federal de Sa o Carlos, Sa o Carlos, Sao Paulo, Brazil, 6 Departamento de Qu mica Fundamental, Instituto de Qu mica, Universidade de Sa o Paulo , Sa o Paulo , Brazil A model betalainic dye was semisynthesized from betanin, the magenta pigment of the red beet, and was effective for livecell 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. - Funding: Funding was provided by the Sao Paulo Research Foundation (FAPESP) (07/00684-6, 10/15042-2, 11/23036-5). LCPG was supported by a FAPESP fellowship (07/59407-1) and the Coordination for the Improvement of Higher Education Personnel (PNPD 427-10/2009) fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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 finally the schizont stage. 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. [8] An essential aspect of the infection process is the remodeling of the RBCM and its protein constituents to permit a higher flux of nutrients and waste products into or away from the intracellular parasite. [9] A single type of broad-specificity channel, variously called the new permeation pathway (NPP), the nutrient channel, or the plasmodial surface anion channel, is responsible for the increased permeability of low molecular weight solutes, both charged and uncharged, with a strong preference for anions. [10]. 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. The functional group attached to the betalainic moiety modulates the amphiphilic character of the dye a (...truncated)


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Letícia Christina Pires Gonçalves, Renata Rosito Tonelli, Piero Bagnaresi, Renato Arruda Mortara, Antonio Gilberto Ferreira, Erick Leite Bastos. A Nature-Inspired Betalainic Probe for Live-Cell Imaging of Plasmodium-Infected Erythrocytes, PLOS ONE, 2013, 1, DOI: 10.1371/journal.pone.0053874