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.
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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)