Abstract
Plant defensins are small cysteine-rich peptides and exhibit antimicrobial activity against a variety of both plant and human pathogens. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, little is known about their activity against protozoa. In a previous study, we isolated a plant defensin named PvD1 from Phaseolus vulgaris (cv. Pérola) seeds, which was seen to be deleterious against different yeast cells and filamentous fungi. It exerted its effects by causing an increase in the endogenous production of ROS (reactive oxygen species) and NO (nitric oxide), plasma membrane permeabilization and the inhibition of medium acidification. In the present study, we investigated whether PvD1 could act against the protozoan Leishmania amazonensis. Our results show that, besides inhibiting the proliferation of L. amazonensis promastigotes, the PvD1 defensin was able to cause cytoplasmic fragmentation, formation of multiple cytoplasmic vacuoles and membrane permeabilization in the cells of this organism. Furthermore, we show, for the first time, that PvD1 defensin was located within the L. amazonensis cells, suggesting the existence of a possible intracellular target.
Highlights
Leishmaniasis is an infectious disease that is prevalent worldwide, in tropical and subtropical regions, killing thousands and debilitating millions of people each year
Cell proliferation assay The effect of PvD1 on the proliferation of L. amazonensis promastigotes was determined by incubating the parasites (1.5×106 parasites/ml) with the PvD1 defensin (300 and 600 μg/ml) for 48 h at 28 ◦C in 200 μl microplate wells
TEM For ultrastructural analyses, untreated or treated L. amazonensis promastigotes were washed with PBS at 37 ◦C and fixed at room temperature in a solution containing 1 % (w/v) glutaraldehyde, 4 % (w/v) paraformaldehyde, 5 mM CaCl2 and 5 % (w/v) sucrose in 0.1 M cacodylate buffer
Summary
Leishmaniasis is an infectious disease that is prevalent worldwide, in tropical and subtropical regions, killing thousands and debilitating millions of people each year. Cell proliferation assay The effect of PvD1 on the proliferation of L. amazonensis promastigotes was determined by incubating the parasites (1.5×106 parasites/ml) with the PvD1 defensin (300 and 600 μg/ml) for 48 h at 28 ◦C in 200 μl microplate wells. At 24 h of the proliferation inhibition assay, L. amazonensis promastigotes were treated with the fluorescent dye Sytox Green which only penetrates cells with structurally compromised plasma membranes.
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