In Vitro Antiplasmodial Activity of Phospholipases A2 and a Phospholipase Homologue Isolated from the Venom of the Snake Bothrops asper

The antimicrobial and antiparasite activity of phospholipase A2 (PLA2) from snakes and bees has been extensively explored. We studied the antiplasmodial effect of the whole venom of the snake Bothrops asper and of two fractions purified by ion-exchange chromatography: one containing catalytically-active phospholipases A2 (PLA2) (fraction V) and another containing a PLA2 homologue devoid of enzymatic activity (fraction VI). The antiplasmodial effect was assessed on in vitro cultures of Plasmodium falciparum. The whole venom of B. asper, as well as its fractions V and VI, were active against the parasite at 0.13 ± 0.01 µg/mL, 1.42 ± 0.56 µg/mL and 22.89 ± 1.22 µg/mL, respectively. Differences in the cytotoxic activity on peripheral blood mononuclear cells between the whole venom and fractions V and VI were observed, fraction V showing higher toxicity than total venom and fraction VI. Regarding toxicity in mice, the whole venom showed the highest lethal effect in comparison to fractions V and VI. These results suggest that B. asper PLA2 and its homologue have antiplasmodial potential.


Introduction
Malaria is responsible for approximately 1.5 million deaths every year in the world. Over 85% of them occur in Africa, with Plasmodium falciparum as the leading species involved in mortality [1,2]. The 2010 WHO report confirmed almost 1 million deaths during the previous year [3]. Malaria is caused by parasites of the genus Plasmodium and is a public health problem in tropical and sub-tropical regions of the world. The most widely used treatment of the clinical syndrome includes artemisinin-based combined therapies [3]. High rates of antimalarial treatment failure have led to the investigation of possible therapeutic alternatives, among which toxins and poisons of animal and plant extracts are included [4][5][6][7][8][9].
Based on the already described antimicrobial and anti-parasitic activity of PLA 2 [17,[25][26][27][28] from snake venoms, the antimalarial potential of the venom of B. asper and PLA 2 s from this venom were explored. Two PLA 2 s from the whole venom were purified and characterized, and their in vitro antiplasmodial activity against P. falciparum was investigated. Cytotoxicity on peripheral blood mononuclear cells (PBMC) and acute toxicity in mice were also evaluated. Results indicate that catalytically-active and inactive PLA 2 s isolated from B. asper venom are cytotoxic against P. falciparum and, thus have the potential as antimalarials.

Isolation of Phospholipase A 2 Fractions
Six fractions obtained by fractionating B. asper venom on ion exchange chromatography on CM-Sephadex C-25 were evaluated for PLA 2 activity. It was found that fraction V was the only positive fraction for PLA 2 activity (see Figure 1A). However, fraction VI, corresponding to a PLA 2 homologue devoid of enzymatic activity (see Section 3.2), was also analyzed for antiplasmodial activity to determine the possibility of catalytically-independent actions. Fractions V and VI were subjected to further separation by RP-HPLC on a C 18 column. This separation revealed that fraction V had four subfractions (see Figure 1B,C), only one of which (V-4) showed PLA 2 activity, whereas fraction VI showed only one peak. These two fractions were used to assess antiplasmodial activity.

Indirect Hemolytic Activity
Fraction V had a minimal indirect hemolytic dose of 1.35 µg, while fraction VI showed no such activity. The PLA 2 isolated by HPLC from fraction V showed a minimum indirect hemolytic dose of 0.82 µg, while the peak obtained by HPLC separation of fraction VI lacked activity (data not shown). The hemolysis test with different substrates (egg yolk, plasma or human serum) yielded similar results in all assays. When indirect hemolytic activity was determined in solution, 100% hemolysis was observed using concentrations of 25 µg/mL for whole venom and 12.5 µg/mL for fraction V, whereas fraction VI lacked PLA 2 activity in all tests (see Figure 2).

Antiplasmodial Activity of the Venom, Fractions and Purified PLA 2 s
Both venom and fractions V and VI exhibit antiplasmodial activity on the FCB1 strain of P. falciparum, with fraction V being more active than fraction VI (see Table 1). On the other hand, the venom was more active than the two fractions evaluated. Guillaume et al. showed that removal of phospholipids from cultures of P. falciparum reduced the antiplasmodial activity of PLA 2 [27], confirming the crucial role of PLA 2 enzymatic activity to control the growth of parasites in this test. Our data demonstrate the antimalarial efficacy of fraction with PLA 2 activity. However, a PLA 2 homologue devoid of enzymatic activity also resulted in restriction of P. falciparum multiplication, confirming a catalytically-independent antiplasmodial activity. This effect could be due to the perturbing action exerted by the PLA 2 homologue in the plasma membrane, thus resulting in an increase in permeability [29]. It has been shown that the C-terminal region of these PLA 2 homologues is responsible for this catalytically-independent membrane perturbation, as demonstrated in bacteria [16,30,31], being, therefore, a different mechanism from the one described for other PLA 2 s [26,27]. The changes observed in the intraerythrocytic development of Plasmodium indicate that structural changes occur, as well as modifications in membrane functions in parasitized red blood cells. In addition, increments and changes in the permeability of the membrane have been described, together with the appearance of new parasite-derived proteins and changes in the composition of membrane lipids [32,33]. The observed increased permeability could also be responsible for the PLA 2 activity on the parasite, as demonstrated by Moll et al., who noted that in the absence of serum in the culture in vitro, PLA 2 lysed parasitized cells [34]. This increase in membrane permeability could also enhance the antimalarial activity of the PLA 2 homologue observed in our experiments.

SDS-PAGE
Electrophoresis showed that proteins of fractions V and VI (lanes 2 and 4, respectively) had molecular weights ranging from 25 kDa and 14 kDa, when fractions were separated in non-reducing conditions, thus evidencing the presence of monomers and dimers, whereas only bands of around 14 kDa were observed (lanes 3 and 5 in Figure 1D, respectively) when these fractions were subjected to reducing conditions, thus corresponding to PLA 2 monomers ( Figure 1D).

Mass Spectrometry and Identification of the Protein
We determined the molecular mass of each of the fractions obtained by RP-HPLC: Fraction V (fractions V-1, V-2, V-3 and V-4) and VI. Mass spectrometric analysis showed that V-1 was of 13786.9 Da, V-2 was of 13950.1 Da, V-3 was of 13972.4 Da, V-4 was of 13974.6 Da and VI was of 13725 Da. The tandem mass MS/MS analysis indicated that the PLA 2 s isolated corresponding to the fractions V-1, V-2, V-3 and VI were K49 PLA 2 homologs, and V-4 was D49 PLA2 (Table 2).      The results of the alignments show that the PLA 2 s and PLA 2 homologues purified from the venom of B. asper from Colombia are similar to other PLA 2 s and PLA 2 homologues present in other Bothrops snakes. In addition, the PLA 2 D49 shows homology with other PLA 2 s from Bothrops, being higher with those of B. asper from Costa Rica (see Figure 5).

Cytotoxic Activity
Analysis of the cytotoxic effect of the whole venom and the different fractions tested showed that fraction V was more cytotoxic than whole venom or fraction VI on PBMC cells (see Figure 8). The cytotoxic activity of venoms and PLA 2 s is a problem in using these in future biomedical applications. However, our results show that the PLA 2 isolated exerts an antimalarial effect at a lower dose than that required to induce cytotoxicity in PBMC and indirect hemolysis.
Other authors have shown that cytotoxic activity is dependent on serum in suspensions of tumor cells and red blood cells [43]. In some experiments, we cultured cells with fetal bovine serum 2% (FBS) and inactivated serum or plasma, and in these conditions, the cytotoxic dose was still higher than the antimalarial dose (results not shown).

Acute Toxicity
The LD 50 of the whole venom of B. asper was 3566 µg/kg (2561 to 3693), whereas no lethality was observed in mice injected with fractions V and VI at doses as high as 15,000 µg/kg (see Table 1).
The envenoming of B. asper induces local and systemic symptoms, such as edema, pain and bleeding, among others, due to the effect of different toxins in the venom, such as PLA 2 , serine proteinases and metalloproteases, among others [19,[44][45][46][47][48][49]. The low toxicity of fraction V and of the PLA 2 homologue isolated from fraction VI compared with the venom indicates their low overall toxicity in mice and reinforces the concept that these fractions are good lead compounds in the search for antimalarial activity. This is in agreement with reports on the use of snake venom PLA 2 s to inhibit microorganisms, such as bacteria and fungi, as well as parasites including Giardia duodenalis, Trypanosoma cruzi, Leishmania spp and P. falciparum [17,30,31,[50][51][52].

Venom and Reagents
The venom was obtained by manual milking of 40 adult specimens from different regions of Colombia held in captivity at the Serpentarium of the University of Antioquia (Medellín, Colombia). Once extracted and pooled, the venoms were centrifuged (3000 rpm, 15 min), and the resulting supernatants were lyophilized and stored at −20 °C until use. Acetonitrile (CH 3 CN) and trifluoroacetic acid (CF 3 COOH) HPLC grade were purchased from Fisher Scientific (Loughborough, UK). Histopaque ® -1077, RPMI-1640 medium culture, Thiazolyl Blue Tretrazolium Bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (Sigma-Aldrich, St Louis, MO, USA). Water for HPLC was deionized to a degree of purity of 17 Ω.

Venom Fractionation
PLA 2s were purified from 50 mg of whole venom of B. asper dissolved in phosphate-buffered saline (PBS), pH 7.2, and passed through a CM-Sephadex C 25 ion exchange column (1.8 × 120 cm) at the flow rate 1.0 mL/min on a low-pressure chromatography system (Econo-System, BioRad, Hercules, CA, USA). The resulting fractions were analyzed for their PLA 2 activity and then PLA 2 positive fractions submitted to a reverse phase HPLC (RP-HPLC) (Shimadzu, Model Prominence, Shimadzu Corporation, Kyoto, Japan) in a C 18 column (pore 5 µm, 250 mm × 4.6 mm mark RESTEK Bellefonte, PA, USA) using a linear gradient (0%-100%) acetonitrile (v/v) in 0.1% (v/v) trifluoroacetic acid at a flow rate 1.0 mL/min. Finally, fractions were lyophilized and stored at −20 °C until use.

Electrophoresis and Molecular Mass Determination
Protein homogeneity of the obtained fractions were determined by electrophoresis under reducing and non-reducing conditions in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 15% [53]. Protein molecular weight was estimated according to a molecular weight markers range of 97.4 to 14.4 kDa (BioRad, Philadelphia, PA, USA). The gels were stained with Coomassie Brilliant Blue G-250. The molecular masses of PLA 2 fractions were confirmed by direct-infusion mass spectrometry in an IonTrap (series 6310, Agilent Technologies, Santa Clara, CA, USA).

Protein Iidentification by HPLC-nESI-MS/MS
The PLA 2 s and PLA 2 homologues isolated from B. asper venom (fractions V and VI see results, Figure

Search Database
Deconvoluted profile spectra were used to search online the MASCOT [55] and Spectrum Mill (Agilent Technology, Santa Clara, CA, USA) in the NCBInr database for protein identification. The parameters of the search included digestion with trypsin and Carbamidomethyilation modified (C) as fixed modification. The minimum score for the intensity of each fraction was 50%, monoisotopic mass, mass tolerance of 2.5 Da and a way to search for identity.

BLAST Search of the Identified Peptides
The identified peptides were subjected to a BLAST search [56] to determine the homology with other PLA 2 family proteins. This homology was performed in BLASTP, the search parameters being non-redundant protein sequence (nr) and a snake organism.

Acute Toxicity of the Venom and Fractions
The Median Lethal Dose (LD 50 ) was determined by the Spearman-Karber method (World Health Organization, 1981) using groups of four mice (Swiss-Webster mice strain) injected intraperitoneally (IP) with varying doses of either fractions or whole B. asper venom, previously dissolved in 0.5 mL PBS, pH 7.2. Fatalities were recorded within 48 h, and the results were expressed as the average of three repetitions.

Cytotoxic Activity
Peripheral blood mononuclear cells (PBMC) were separated by centrifugation of citrated human blood (400g, 30 min) with Histopaque ® -1077 (Sigma-Aldrich, St Louis, MO, USA), washed with PBS and transferred to 96 well plates at a concentration of 3 × 10 5 cells/well. Cells were cultured with different concentrations of fractions (37 °C, 5% CO 2 ) for 24 h. After this time, 40 µL of MTT was added and incubated for 3 h (same conditions as described). The reaction was halted by adding 130 µL of dimethyl sulfoxide (DMSO) and readings were performed in a microplate reader at 420 nm. The 50% cytotoxic dose was calculated by linear regression [57].

Indirect Hemolysis
This was evaluated following the method that uses agarose gel-erythrocyte-egg yolk [58,59]. We estimated the minimum indirect hemolytic dose (MIHD), defined as the dose of venom producing a hemolytic halo of 20 mm in diameter after 20 h. In addition, indirect hemolytic activity was assessed on red blood cells in suspension. For this, different doses of either the whole venom or fractions V and VI were incubated with fresh human red blood cells for 30 min at 37 °C in the presence of 250 µL of inactivated human serum, inactivated human plasma, egg yolk or PBS. Afterwards, samples were centrifuged, and the percentage of lysis was determined by recording the absorbance at 540 nm as an index of released hemoglobin. As a control of 100% hemolysis, 2%Triton X-100 was used. The results were expressed as percentage of lysis, and the venom or toxin concentration producing 100% hemolysis was determined.

Cultivation of Plasmodium falciparum
Based on the procedure described by Trager and Jensen [60], parasites were grown at 37 °C in A+ human erythrocytes to a hematocrit of 2% and 3%-6% parasitemia under an atmosphere of 3% CO 2 , 6% O 2 and 91% N 2 .

Determination of Percentage of Growth Inhibition of P. falciparum by B. asper PLA 2 Fractions
Increasing concentrations of PLA 2 fractions V and VI in complete medium were plated in 96-well plates (100 µL/well) and incubated with asynchronous P. falciparum FCB1 (1.5% parasitemia, 4% hematocrit, 100 µL/well). Parasites were incubated as previously described [60]. After 24 h, 0.5 mCi of 3 H-hypoxanthine was added to the culture, and parasites were cultured for further 24 h at the same conditions. Finally, the plates were freeze-thawed, and parasites were harvested onto filter paper, added to liquid scintillation cocktail and the incorporation of 3 H-hypoxanthine determined in a Microbeta counter 1450 (Wallac, Perkin Elmer, Waltham, MA, USA).
The percentage of growth inhibition was calculated based on 100% uptake of the 3H-hypoxanthine of controls (parasites in culture medium, incomplete RPMI). Growth inhibition was calculated based on 100% uptake of the 3H-hypoxanthine control in parasites in the absence of PLA 2 s or PLA 2 homologues. The IC 50 values correspond to the venom or toxin concentration required to kill 50% of the parasites within 48 h, and was determined from dose-response curves according to Desjardins et al. [58].

Statistical Analysis
The results are presented as mean ± S.E.M of three replicates, and experimental differences between means were determined by analysis of variance followed by Dunnett's test for intragroup comparisons. Significance was set up at p < 0.05.

Conclusions
Our observations suggest that PLA 2 s and PLA 2 homologues present in the venom of Bothrops asper represent promising lead compounds in the search for novel antimalarial agents. Further studies should be performed on the identification of the molecular determinants of this activity.