Calcium Ionophore, Calcimycin, Kills Leishmania Promastigotes by Activating Parasite Nitric Oxide Synthase

Leishmaniasis is an infectious disease caused by protozoan parasites of the genus Leishmania. There is no vaccine against human leishmaniasis and the treatment of the disease would benefit from a broader spectrum and a higher efficacy of leishmanicidal compounds. We analyzed the leishmanicidal activity and the mechanism of action of the calcium ionophore, calcimycin. L. major promastigotes were coincubated with calcimycin and the viability of the cells was assessed using resazurin assay. Calcimycin displayed dose-dependent effect with IC50 = 0.16 μM. Analysis of propidium iodide/LDS-751 stained promastigotes revealed that lower concentrations of calcimycin had cytostatic effect and higher concentrations had cytotoxic effect. To establish the mechanism of action of calcimycin, which is known to stimulate activity of mammalian constitutive nitric oxide synthase (NOS), we coincubated L. major promastigotes with calcimycin and selective NOS inhibitors ARL-17477 or L-NNA. Addition of these inhibitors substantially decreased the toxicity of calcimycin to Leishmania promastigotes. In doing so, we demonstrated for the first time that calcimycin has a direct leishmanicidal effect on L. major promastigotes. Also, we showed that Leishmania constitutive Ca2+/calmodulin-dependent nitric oxide synthase is involved in the parasite cell death. These data suggest activation of Leishmania nitric oxide synthase as a new therapeutic approach.


Introduction
Leishmania is the genus of protozoan parasites which cause leishmaniasis. The reservoir hosts of most Leishmania species from which parasites are transmitted to humans by phlebotomine sand flies are rodents and canids (zoonotic leishmaniasis); transmission from infected to noninfected humans occurs only in some Leishmania species (anthroponotic leishmaniasis) (reviewed in [1]). In some cases, parasites can be transmitted from infected to noninfected humans also through needle sharing among intravenous drug users [2] and by organ transplantation and blood transfusion (reviewed in [3]). Congenital transmission from mother to child was also described [4]. There are 12 million people infected with Leishmania and 350 million people at risk of infection in 98 countries [5]. Recent refugee crisis caused a devastating outbreak of leishmaniasis in the Middle East and North Africa [6]. This outbreak and the global warming are the main factors promoting the spread of leishmaniasis to Europe [7][8][9] and to North America [10].
Despite its huge impact on the populations in vast areas, leishmaniasis is one of the most neglected diseases. Up to date, no effective vaccine against human leishmaniasis has been developed [11,12]. The spectrum and efficacy of available antileishmanial drugs are also limited (reviewed in [13]). The first-line drugs, including amphotericin B and pentavalent antimonials, have serious side effects [13]. Thus, there is a continuing need for new targets for antileishmanial therapy and new chemical substances with leishmanicidal effect.

BioMed Research International
Moreover, pretreatment of macrophages by calcimycin before their infection with Leishmania major led to a decrease of 3 [H]-thymidine incorporation by intracellular amastigotes [22]. Yet, there have been no data about the effect of calcimycin on Leishmania promastigotes and no data about the mechanism of action of calcimycin.
We therefore examined the antipromastigote activity of calcimycin and described its mode of action by showing that it activates parasite's nitric oxide synthase.

Promastigote Growth Inhibition
Assay. Leishmania promastigotes in the logarithmic phase of growth were seeded into black 384-well plates (cat. number 3571, Corning, New York, NY) at a density of 15,000 parasites/25 l/well in supplemented Schneider's Insect Medium using the Multidrop Combi (Thermo Fisher Scientific, Waltham, MA). Immediately after plating Leishmania promastigotes, calcimycin and the reference compound amphotericin B, which is currently the best option for treatment of visceral leishmaniasis [9], were added in a range of concentrations and incubated for 48 hours at 23 ∘ C. The time of incubation was chosen as one with proven efficiency in previous experiments by others [24]. The assays were performed in triplicate. The metabolic capacity of the parasites was measured after 2.5 h coincubation with the CellTiter-Blue5 Reagent (cat. number G8082, Promega, Madison, WI) using EnVision Plate Reader (PerkinElmer, Waltham, MA). The data were fitted using nonlinear regression (exponential, one-phase decay). Half maximal inhibitory concentration (IC 50 ) was calculated as a concentration of the compounds, at which the viability of the parasites was 50%.

Leishmania Promastigote Counts.
After 48-hour incubation with 0.5, 1.0, and 2.0 M calcimycin or without calcimycin, L. major promastigotes were counted in duplicates with Z2 COULTER COUNTER (Beckman Coulter, Inc., Brea, CA). The aforementioned concentrations of calcimycin were selected based on the results of promastigote growth inhibition assay (Figure 1(a)), representing submaximal and maximal leishmanicidal effect. For counting, 50 l of Leishmania parasite suspensions was diluted in 20 ml of ISOTON II Diluent (cat. number 8546719, Beckman Coulter, Inc.).

Analysis of Cell Death of L. major Promastigotes.
After 48hour incubation with 0.5, 1.0, and 2.0 M calcimycin, L. major promastigotes were washed and incubated with propidium iodide (PI) (cat. number P4170, Sigma) in phosphate-buffered saline (PBS) for 10 minutes in the dark at room temperature. After that, laser dye styryl-(LDS-) 751 (Invitrogen, Carlsbad, CA) was added and the samples were incubated for additional 20 minutes under the same conditions. Then the samples were washed, resuspended in PBS, and analyzed for fluorescence using flow cytometer FACSCalibur (Becton, Dickinson and Company). PI is a dye staining necrotic and late apoptotic cells with a penetrable plasma membrane. PI fluorescence was detected in both orange (FL2 channel, 585 ± 21 nm bandpass filter) and red (FL3 channel, >670 nm longpass filter) ranges of the spectrum. With our instrument settings, PI positive cells generate an average angle of 45 degrees between -axis (FL2 channel) and -axis (FL3 channel). LDS-751 is a cell permeable dye, which is accumulated in polarized mitochondria. Thus, LDS-751 stains nonapoptotic cells and does not stain the early and late apoptotic or necrotic cells. LDS-751 fluorescence was measured in the red (FL3 channel, >670 nm longpass filter) part of the spectrum. The samples were also analyzed by light scatter. For each sample, 10,000 events were acquired.

Promastigote Growth Is Inhibited by Calcimycin and
Amphotericin B. The assay of metabolic capacity of parasites demonstrated that both calcimycin and the reference compound amphotericin B are highly active against L. major promastigotes (Figure 1(a)) with IC 50 of 0.159 M and 0.0614 M, respectively. The direct counting by Z2 COULTER COUNTER showed that, in cultures treated with calcimycin, the number of L. major promastigotes was drastically decreased in comparison with untreated cultures. The original cultures were incubated for 48 hours before treatment and adjusted to the working concentration of 0.6 × 10 6 /ml. The cultures of multiplying parasites have high growth potential and after additional 48 hours of cultivation, the concentration of parasites in untreated cultures rises 28 times, to 17.32 × 10 6 /ml, whereas in cultures grown with 0.5 M, 1.0 M, and 2.0 M calcimycin, concentrations of promastigotes were dramatically lower: 8.46 × 10 6 /ml, 5.6 × 10 6 /ml, and 4.9 × 10 6 /ml, respectively (Figure 1(b)).

Calcimycin Has Direct Cytotoxic Effect on Leishmania
Promastigotes. To test whether calcimycin effect was cytostatic or cytotoxic, we studied parasite staining with PI and LDS-751.

Discussion
In the present study, we demonstrated for the first time that calcimycin has a direct dose-dependent leishmanicidal effect on L. major promastigotes (Figure 1). Lower doses of calcimycin inhibit Leishmania growth but do not augment cell death in Leishmania promastigotes, whereas higher doses of calcimycin cause promastigote death accompanied by the loss of mitochondrial polarization and plasma membrane integrity ( Figure 2). Next, we investigated the mechanism of action of calcimycin on L. major. It is known that, in mammalian cells, calcimycin causes hyperactivation of constitutive Ca2 + /calmodulin-dependent NOS [17,18]. At the same time, NOS overexpression in mammalian cells was shown to be associated with the impairment of mitochondrial function and apoptosis [25]. As in calcimycin-treated Leishmania, we also observed the loss of mitochondrial potential ( Figure 2); we proposed that calcimycin causes Leishmania  100%). Data are presented as mean ± SD (three independent experiments; each assay was performed in triplicate). Promastigote survival was compared using Mann-Whitney test. * * corresponds to ≤ 0.01; * * * corresponds to ≤ 0.001. apoptosis via hyperactivation of NOS. To test this hypothesis, we investigated the ability of specific inhibitors of NOS, N -Nitro-L-arginine (L-NNA) and ARL-17477, to attenuate the leishmanicidal effect of calcimycin. It should be mentioned that at least N -Nitro-L-arginine methyl ester was already shown to inhibit not only mammalian neuronal NOS [26] but also constitutive NOS of L. amazonensis amastigotes [20]. Indeed, our experiments demonstrated that L-NNA as well as ARL-17477 was able to block antileishmanial effect of calcimycin ( Figure 3). This finding supports the hypothesis that killing of Leishmania parasites is mediated by hyperactivation of constitutive NOS stimulated by calcimycin.
Our results extend the data about effect of calcimycin on Leishmania parasites obtained by others using different experimental designs [21,22]. In their pioneering work, Buchmuller-Rouiller and Mauel [21] infected bone marrowderived macrophages of CBA mice with L. enriettii, incubated them for 4 hours with calcimycin (0.01-0.25 M) and with or without LPS, washed the cultures, and continued incubation in presence or absence of LPS for additional 20 hours. Parasite survival was measured by [ 3 H]-thymidine incorporation into L. enrietti released from SDS-lysed macrophages. It was found that priming with calcimycin led to decrease of [ 3 H]thymidine incorporation only in presence of LPS. However, the absence of the effect of calcimycin alone might be attributed to the low concentration of calcimycin and to the very short exposure to the compound. Lanza and coworkers [22] pretreated peritoneal macrophages of BALB/c mice for 48 hours with 1 M calcimycin, washed the cells, and infected them for 3 hours with L. major, and [ 3 H]-thymidine was added for 18 hours. This pretreatment led to a decrease of [ 3 H]-thymidine incorporation by Leishmania amastigotes in macrophages. However, this experimental setting does not allow distinguishing if calcimycin stimulates macrophages to kill Leishmania or if it has cytostatic or cytotoxic effect on its own.

Conclusions
This is the first study demonstrating that calcimycin has direct cytostatic and cytotoxic effect on Leishmania promastigotes. We demonstrate that calcimycin-induced Leishmania cell death is accompanied by the loss of mitochondrial polarization and plasma membrane integrity and can be blocked by specific inhibitors of constitutive Ca2 + /calmodulindependent nitric oxide synthase. The data imply that leishmanial nitric oxide synthase is crucial for the antileishmanial effect of calcimycin and suggest hyperactivation of leishmanial nitric oxide synthase as a new therapeutic approach.

Disclosure
This publication is based on a part of the Ph.D. thesis of Dr. Igor Grekov [27]. The funders had no role in study design, data collection and analysis, or decision to publish or preparation of the manuscript.

Conflicts of Interest
The authors declare that they have no financial conflicts of interest in this investigation.