PvD₁ defensin, a plant antimicrobial peptide with inhibitory activity against Leishmania amazonensis

Leishmaniasis is an infectious disease that is prevalent worldwide, particularly in tropical and subtropical regions, killing thousands and debilitating millions of people each year. With 2 million new cases reported annually, infection by the Leishmania parasite represents an important global health problem for which there is no vaccine and few effective drugs [1].

The selection of an increasing number of antibiotic-resistant micro-organisms and other antimicrobial agents has attracted the attention of many researchers in an attempt to develop new therapeutic agents [2]. The therapeutic potential of AMPs (antimicrobial peptides) is enhanced owing to the ability of these compounds to rapidly kill a large number of micro-organisms such as bacteria, viruses, fungi and parasites that are multidrug-resistant [3,4]. There is an increasing interest in the study of AMPs because of their capacity to interact with certain cellular membranes, and the resulting antimicrobial activity that they display against pathogens [5].

Promising AMPs include the plant-derived defensins, a family of basic peptides which consists of 45–54 amino acids, arranged in three-dimensional structures formed by three antiparallel β-strands and one α-helix. This structure is stabilized by four disulfide bonds, which form a cysteine-stabilized α-helix β-strands motif, commonly found in these peptides [5,6]. The antimicrobial activity of plant defensins is mainly observed against fungi. However, some bacteria, particularly Gram-positive species, are also inhibited, although the activity is less pronounced than that against fungi. The growth of several fungal species, including several filamentous fungi and yeast cells, was inhibited when incubated with these peptides [7–21]. Despite the broad inhibitory activity that plant defensins exhibit against different micro-organisms, scientists know little about their activity against protozoa.

In a previous study, we isolated a plant defensin named PvD₁ from Phaseolus vulgaris (cv. Pérola) seeds. This protein was able to negatively affect different yeast cells and filamentous fungi [22], 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 [23]. In the present study, we investigated the action of PvD₁ against the protozoan Leishmania amazonensis. We were also interested to understand the mechanism by which PvD₁ affects L. amazonensis promastigotes.

P. vulgaris L. seeds were supplied by the Empresa de Pesquisa Agropecuária do Estado do Rio de Janeiro (Pesagro), Campos dos Goytacazes, Rio de Janeiro, Brazil.

Promastigote-stage L. amazonensis (Josefa strain) were supplied by Laboratório de Biologia Tecidual, Centro de Ciências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil. The protozoa were cultivated in 5 ml of Warren's medium [90% brain heart broth (Fluka) containing 10% (v/v) heat-inactivated FBS], enriched with 0.01% folic acid and 0.4% haemin at 28°C. The protozoa were transferred to new medium every 3 days.

The purification of the defensin from P. vulgaris (cv. Pérola) seeds was conducted as described by Games et al. [22].

PvD₁ defensin purification was monitored by SDS/Tricine gel electrophoresis performed according to the method previously described by Schägger and Von Jagow [24].

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. The peptide was diluted in DMSO (1.5% final concentration) and Warren's medium (100 μl) and incubated with the parasites in Warren's medium (100 μl). The proliferation of the parasites was monitored at 24 and 48 h by cell counting in Neubauer chamber. The proliferation of L. amazonensis promastigote controls (without the addition of peptides) was also assessed. The experiments were performed in triplicate, and S.E.M. were calculated.

Membrane permeabilization of L. amazonensis promastigotes was assessed by measuring Sytox Green uptake as described previously, with some modifications, by Thevissen et al. [25]. Sytox Green is a dye that only penetrates cells when the plasma membrane is structurally compromised. Once inside the parasitic cytoplasm, it binds to nucleic acids, resulting in a fluorescent complex. The L. amazonensis promastigotes (1.5×10⁶ parasites/ml) were incubated with PvD₁ at a concentration of 300 μg/ml for 24 h at 28°C in 200 μl microplate wells. Aliquots (100 μl) of the parasite cell suspension were incubated with 0.2 μM Sytox Green in 1.5 ml microcentrifuge tubes for 30 min at 25°C with periodic agitation. After incubation, the parasites were fixed in 2% (w/v) paraformaldehyde. The cells were examined using a DIC (differential interference contrast) microscope (Axiophoto, Zeiss) equipped with a fluorescence filter set for the detection of fluorescein (excitation wavelengths, 450–490 nm; emission wavelength, 500 nm). Negative (no PvD₁ added) controls were also run to evaluate baseline membrane permeability.

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 (pH 7.2). The cells were post-fixed for 1 h in a solution containing 2% OsO₄, 0.8% potassium ferrocyanide and 5 mM CaCl₂ in 0.1 M cacodylate buffer (pH 7.2), rinsed with 0.1 M cacodylate buffer (pH 7.2), dehydrated in acetone and embedded in PolyBed (Polysciences). Thin sections were stained with uranyl acetate and lead citrate and subsequently examined using a Zeiss 900 Transmission Electron Microscope at 80 kV acceleration.

PvD₁ was conjugated to FITC (Sigma), according to the manufacturer's instructions. The L. amazonensis promastigotes (1.5×10⁶ parasites/ml) were incubated with PvD₁–FITC at a concentration of 150 μg/ml for 24 h at 28°C in 200 μl microplate wells. The cells were examined using a DIC microscope equipped with a fluorescence filter set for fluorescein detection (excitation wavelengths, 450–490 nm; emission wavelength, 500 nm).

The effect of PvD₁ on the proliferation of L. amazonensis promastigotes was evaluated. Figure 1 shows the inhibitory effects of PvD₁ (at concentrations of 300 and 600 μg/ml) towards the proliferation of parasites for periods of 24 h and 48 h. When compared with the control parasites grown in the absence of PvD₁, inhibitory rates of 70 and 89% were observed when testing PvD₁ at a concentration of 300 μg/ml at 24 h and 48 h respectively, whereas at a concentration of 600 μg/ml, inhibition rates of 87% and 96.5% were observed at 24 h and 48 h respectively.

In order to investigate further the negative effects of PvD₁ on L. amazonensis promastigote proliferation, we analysed whether cell membranes had been disrupted by the defensin. 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. The observation of these cells by fluorescence microscopy showed that the L. amazonensis promastigotes were labelled by Sytox Green when treated with 300 μg/ml PvD₁ (Figure 2). In control cells, when parasites were grown in the absence of PvD₁, no fluorescence was observed.

To confirm the effect of PvD₁ defensin on membrane permeabilization of L. amazonensis promastigotes, these cells were analysed by TEM. The ultrastructure analysis of promastigotes treated with 300 μg/ml PvD₁ revealed that this defensin was able to cause plasma membrane disruption followed by leakage of cytoplasmic material. It also caused cytoplasmic fragmentation and vacuolization. In contrast, control cells exhibited membrane integrity and normal intracellular organization (Figure 3).

In order to investigate further whether the PvD₁ defensin could be internalized in L. amazonensis promastigotes, its ability to penetrate and accumulate inside the cell was evaluated by coupling the peptide with FITC. After the L. amazonensis promastigote proliferation inhibition assay using 150 μg/ml PvD₁–FITC, we observed the presence of PvD₁–FITC in intracellular spaces of these cells (Figure 4), suggesting a possible intracellular target of PvD₁ in L. amazonensis promastigotes. In control cells, no fluorescence was observed.

Plant defensins are capable of inhibiting the growth of a wide variety of filamentous fungi and yeast [5,6,23,22,26,27]. Despite the wide inhibitory activity that plant defensins have against different micro-organisms, there is a gap in our knowledge regarding the mechanism of action of plant defensins against protozoa. Only two studies to date show plant defensin activity against protozoan parasites of the genus Leishmania [28,29]. In the present study, we analysed the activity of a plant defensin, PvD₁, against L. amazonensis promastigotes and characterized some aspects of this plant defensin's action against this parasite.

We observed that at 300 μg/ml, PvD₁ was able to inhibit 70% of the protozoan proliferation, after 24 h of incubation (Figure 1). Berrocal-Lobo et al. [30] observed the activity of a PTH1 defensin from Solanum tuberosum and a thionin from Triticum aestivum against L. donovani; however, these authors did not test these peptides against L. amazonensis. More recently, Souza et al. [29] showed that the natural defensin from Vigna unguiculata seeds (Vu-Def), as well as its recombinant homologues (Vu-Defr), both at a concentration of 100 μg/ml, were also able to inhibit the proliferation of the parasite L. amazonensis by approximately 50% after 48 h incubation. Nonetheless, it is still unknown whether the plant defensins would affect other species of protozoa with similar strength.

After treatment of L. amazonensis promastigotes with PvD₁, we used the Sytox Green fluorescent dye which binds to nucleic acids when the cell plasma membranes are structurally compromised. We observed that PvD₁ was capable of causing damage to the plasma membrane (Figure 2), which was confirmed by the effects of PvD₁ on membrane permeabilization, seen using TEM. Using ultrastructural analysis, we showed that the cells of protozoa treated with the defensin exhibited a disruption of the promastigote plasma membrane leading to the loss of cytoplasmic material as well as cytoplasmic fragmentation and formation of multiple cytoplasmic vacuoles (Figure 3). Bera et al. [28] examined the effects of the AMP indolicidin (derived from bovine neutrophils) and two other peptides, 27RP and SPFK (derived from bovine seminal plasma), on Leishmania donovani. Interestingly, cells treated with these peptides exhibited extensive degeneration of intracellular organization and the formation of multiple cytoplasmic vacuoles without disruption of the plasma membrane. In contrast with the effect caused by AMPs, cells treated with amphotericin B exhibited no vacuolarization, although amphotericin B causes significant disruption of the plasma membrane. In the present study, we found both effects for PvD₁, which is the first reported plant AMP capable of causing cytoplasm fragmentation, formation of multiple cytoplasmic vacuoles and plasma membrane disruption of L. amazonensis promastigotes.

After this primary membrane-permeabilizing event, we analysed whether PvD₁ was able to internalize L. amazonensis promastigotes. For this, FITC-tagged PvD₁ was observed by fluorescence microscopy (Figure 4). The present study is the first to show that a plant defensin was able to enter L. amazonensis cells. On the basis of these data, we suggest the existence of a possible intracellular target for this defensin as a part of the mechanism responsible for leading to the protozoan's death.

In the present paper, we report the activity of a plant defensin against L. amazonensis and provide information on its mechanism of action. We demonstrate that, in addition to 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 these cells. Furthermore, we show for the first time that PvD₁ defensin located within these cells may be acting on a possible intracellular target. These results open new perspectives regarding the antimicrobial mechanism of plant defensins as it suggests that the toxicity of these peptides may not be restricted to the plasma membrane.

The study was conceived by Valdirene M. Gomes and Edésio J.T. de Melo. Experimental procedures were carried out by Érica de O. Mello, Viviane V. do Nascimento and Laís P. Carvalho. Data analyses were performed by Érica de O. Mello, Viviane V. do Nascimento, Laís P. Carvalho, André de O. Carvalho, Valdirene M. Gomes and Edésio J.T. de Melo. The paper was written by Érica de O. Mello, Viviane V. do Nascimento, André de O. Carvalho, Katia V.S. Fernandes and Valdirene M. Gomes.

This study comprises part of the postdoctoral research of Dr Viviane Veiga do Nascimento and part of the D.Sc. degree thesis of Erica O. Mello. This work was performed at the Universidade Estadual do Norte Fluminense Darcy Ribeiro. We are grateful to L.C.D. Souza and Kokis V.M. for general laboratory technical support.

We acknowledge the financial support of the Brazilian agencies Programa Nacional de Pós Doutorado (PNPD)/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CAPES/Toxinology, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).

AMP

antimicrobial peptide

DIC

differential interference contrast