Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes

Experimental Parasitology 113 (2006) 186–192 www.elsevier.com/locate/yexpr

Activity of an engineered synthetic killer peptide on Leishmania major and Leishmania infantum promastigotes Dianella Savoia a,¤, Sara Scutera a, Stefania Raimondo a, Stefania Conti b, Walter Magliani b, Luciano Polonelli b a

Department of Clinical and Biological Sciences, University of Torino, at S. Luigi Gonzaga Hospital, Regione Gonzole 10, 10143 Orbassano (To), Italy b Department of Pathology and Laboratory Medicine, University of Parma, V. Gramsci 14, 43100 Parma, Italy Received 2 September 2005; received in revised form 5 January 2006; accepted 6 January 2006 Available online 17 February 2006

Abstract This study was undertaken to analyze the eVect of an engineered, killer decapeptide (KP) on Leishmania major and Leishmania infantum promastigotes. The KP was synthesized on the basis of the sequence of a recombinant, single-chain anti-idiotypic antibody acting as a functional internal image of a yeast killer toxin. The evaluation of in vitro inhibitory activity of KP on L. major and L. infantum, release of intracellular green Xuorescent protein (GFP) molecules by L. major, DNA fragmentation, and ultrastructural analysis (TEM) of L. infantum upon KP treatment were performed. KP presented antiproliferative and leishmanicidal activity with LC50/1 day of 58 and 72 M for L. major and L. infantum, respectively. A dose-dependent decrease in proliferation and increase of killing of promastigotes was seen after KP treatment. No DNA fragmentation in L. infantum promastigotes or release of intracellular GFP molecules on peptide treatment of a GFP expressing L. major clone was demonstrated. Moreover the plasma-membrane was not disrupted, but, by TEM analysis, intracellular damage was observed. © 2006 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: L. major; L. infantum; Killer peptide; DNA fragmentation; Electron microscopy; DNA, deoxyribonucleic acid; KP, killer peptide; GFP, green Xuorescent protein; TEM, transmission electron microscopy

1. Introduction Leishmania is a protozoan parasite that causes various serious diseases threatening millions of people worldwide (WHO, 2005), particularly in developing countries. The infections, transmitted via the bite of phlebotomine sandXies, cause cutaneous, mucocutaneous, or visceral leishmaniasis (VL). In particular, VL is considered an emerging zoonosis in southern Europe, mainly as an opportunistic infection in AIDS, neoplasia, and transplant patients (Desjeux and Alvar, 2003; Marty et al., 1994). In the Mediterranean basin, Leishmania infantum is an important parasite in AIDS patients; up to 9% of these subjects suVer from newly

*

Corresponding author. Fax: +30 0119038639. E-mail address: [email protected] (D. Savoia).

0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2006.01.002

acquired or reactivated VL (Morales et al., 2002). Treatment relies primarily on chemotherapy: Wrst-line drugs, such as pentavalent antimonials, and second-line drugs, such as amphotericin B, liposomal amphotericin B, and pentamidine, are toxic and too expensive for developing countries. Furthermore, low dosage schemes and poor compliance have led to an increasing resistance to pentavalent antimonials (Ouellette et al., 2004). Even if, in the last few years, new drugs such as miltefosine and paromomycin have been developed (Croft et al., 2005), there remains a need to develop new tools and strategies for this neglected disease. Recently, a monoclonal antibody (KTmAb) mimicking a yeast killer toxin (KT) produced by the yeast Pichia anomala has shown in vitro leishmanicidal activity (Savoia et al., 2002). KTmAb, as well as KT and KT-like polyclonal (KTAbs) and recombinant (KTscFv) microbicidal antibodies, produced by

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idiotypic vaccination with a KT neutralizing mAb (Magliani et al., 1997; Polonelli et al., 1994, 1997), showed in vivo and/or in vitro microbicidal activity against a broad range of taxonomically unrelated prokaryotic and eukaryotic pathogens (Conti et al., 1998, 2002; Séguy et al., 1998). The linkage to speciWc KT receptors (KTR), which have been putatively considered in sensitive microorganisms to be cell wall -glucans (Cenci et al., 2004; Guyard et al., 2002), is a crucial step in the microbicidal eVect of these molecules. Interestingly, a killer decapeptide (KP) synthesized and engineered on the basis of the KTscFv sequence exerted a strong in vitro and in vivo microbicidal activity against diVerent fungal pathogens, such as Candida albicans, Cryptococcus neoformans, and Paracoccidioides brasiliensis (Cenci et al., 2004; Polonelli et al., 2003; Travassos et al., 2004). In competition assays, it was demonstrated (Polonelli et al., 2003) that laminarin, a soluble 1,3glucan, completely abolishes the activity of KP, suggesting that KP linkage to 1,3-glucan is a crucial step in the antifungal eVect of this molecule interfering with the formation of yeast cell wall. The aim of the current study was to evaluate the in vitro activity of KP against L. major and L. infantum in comparison with a scrambled irrelevant decapeptide. Potential targets of KP in the protozoan cells have been investigated by Xuorescence, DNA electrophoresis, and transmission electron microscopy; additionally the leishmanicidal activity of laminarinase is reported. 2. Materials and methods 2.1. Chemicals and peptides Laminarinase, a 1,3--D-glucan 3(4)-glucanohydrolase, was obtained from Sigma–Aldrich (Milano, Italy). Synthesis and optimization through alanine scanning of KP (AKVTMTCSAS) have been described in detail elsewhere (Polonelli et al., 2003). Heat-inactivated (100 °C, 10 min) laminarinase and a scrambled peptide (SP, MSTAVSKCAT), containing the same amino acids of KP in a diVerent sequence, were included as negative controls. 2.2. Parasites Leishmania major (strain MHOM/IL/67/JERICHO-II) and Leishmania infantum (strain MHOM/TN/80/IPT1) promastigotes were maintained in vitro at 25 °C in modiWed Tobie’s diphasic medium (Taylor and Evans, 1978). Before use, promastigotes were grown in “complete medium,” constituted by 199 medium (Invitrogen, CA) supplemented with 20% heat-inactivated FCS (Invitrogen), 100 U/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 40 mM Hepes, 0.1 mM adenine (in 50 mM Hepes), 5 g/ml hemin (in 50% triethanolamine), and 1 g/ml 6-biotin (in 95% ethanol). The parasites were cultured at 25 °C for 5 days to reach the stationary (more virulent) phase of growth, unless otherwise stated. They were then collected by centrifugation (1400g, 5 min at room temperature), washed in saline

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solution, and resuspended in fresh complete medium to a Wnal concentration of 2 £ 105 viable promastigotes/ml. The number of organisms was determined by counting with a hemocytometer (Thoma chamber) after vital staining with trypan blue (dye exclusion method). Some experiments were performed by using L. major promastigotes expressing green Xuorescent protein (GFP) as a Xuorescent marker. The pXG-GFP+ (strain B2799) construct (Ha et al., 1996), a kind gift of Dr. S.M. Beverley (Washington University School of Medicine, St. Louis), was cloned into competent Escherichia coli Top 10 (Invitrogen, Carlsbad, CA) and selection was eVected on Luria Bertani (LB) agar containing ampicillin (100 g/ml). The construct was introduced into L. major promastigotes by electroporation (BioRad Gene Pulsar, USA) according to a technique previously described (Seay et al., 1996). The transfectant clone was selected on complete medium containing progressively increasing drug levels of G418 (Sigma, Milan, Italy) and therefore maintained in the same medium with 20 g/ml G418. Fluorescence, detected by UV microscopy (Laborlux, Leitz) at 520 nm and distributed throughout most of the cell, revealed GFP-expressing L. major. 2.3. In vitro anti-leishmanial activity The eVect of the diVerent reagents on L. major and L. infantum promastigotes was assessed by a method similar to that of Savoia et al. (2004). Promastigotes (2 £ 105 viable cells/ml) were incubated in complete medium in the presence of 25, 50, 100, and 200 g/ml of the peptides. In selected experiments laminarinase (75, 150, and 300 g/ml) or heat (100 °C for 10 min)-inactivated laminarinase was also assessed. After 24 and 48 h incubation at 25 °C with shaking, parasite survival was estimated by dye exclusion to identify viable promastigotes in the cultures. At the end of every incubation time, reversibility was assessed by adding complete fresh medium to the cultures at a 10:1 ratio and viability was reassessed after a further 24 and 48 h incubation. The experiments were performed in triplicate, and the results (means of the samples at each point) are reported as percentage of killing of promastigotes. The concentration of KP which caused a 50% reduction in survival or viability (LC50) in comparison to that in an identical culture without the compound was evaluated after 24 h. This value was determined by nonlinear regression analysis, by plotting the number of viable promastigotes versus log KP concentration by use of GraphPad Prism 3 software. 2.4. Methylene blue dye exclusion assay The eVect of KP (200 g/ml) on the uptake of methylene blue dye by L. major promastigotes was assessed according to the technique reported by Hammer et al. (2004). Control and treated parasites were incubated at 25 °C with shaking and samples were taken at 1, 2, and 4 h. Each sample of 80 l was added to 20 l of 0.05% methylene blue (w/v prepared in sterile distilled water), mixed and left for 5 min

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at room temperature. Cells were examined microscopically (400£) and the percentage of stained promastigotes calculated.

3. Results

2.5. Measurement of GFP Xuorescence

The dose-dependent activity of KP against L. major and L. infantum promastigotes is shown in Figs. 1A and B. Standard deviations were 610% of the values reported. At a KP concentration of 100 g/ml, growth was totally inhibited after 48 h of contact, whereas concentrations of 25 g/ ml were nearly ineVective (data not shown). Laminarinase (150 g/ml) reduced the growth of promastigotes, in particular of L. major, similarly to KP; less inhibition was observed with 75 g/ml, but it was slightly greater with 300 g/ml (data not shown). No activity was seen with the irrelevant decapeptide SP or with heat-inactivated laminarinase, both having growth curves similar to control in medium (data not shown). The eVect of KP was irreversible as assessed by adding fresh medium to the cultures at the end of incubation. The reduction in the growth (Fig. 1) and the increase of L. major and L. infantum killing strongly suggest a leishmanicidal activity of KP. The LC50 values, obtained after 24 h treatment, were 58 g/ml (58 M) for L. major and 72 g/ml (72 M) for L. infantum.

A four day culture (late log phase of growth) of L. major promastigotes expressing GFP was diluted in phosphatebuVered saline (PBS) supplemented with 0.1% glucose to obtain a 106 cells/ml suspension. Promastigotes were treated for 3 h at 25 °C with KP or SP (100 and 200 g/ml) and with the polyene drug amphotericin B (0.1 and 0.2 g/ ml), similar to a procedure described by Bera et al. (2003). The same test was conducted with a control culture. After centrifugation (3000 rpm, 10 min), the supernatant of the diVerent preparations was collected and GFP Xuorescence signals were measured in a Xuorimeter (Kontron, Toronto, Canada) at excitation wavelength 475 nm and emission wavelength 535 nm. 2.6. DNA fragmentation assay by agarose gel electrophoresis Qualitative analysis of DNA fragmentation was performed (two independent experiments) by agarose gel electrophoresis, as previously described (Paris et al., 2004). The genomic DNA was extracted from untreated and KP (100 and 200 g/ml)- or miltefosine (25 M)-treated 107 L. infantum promastigotes; after lysis and digestion, DNA (10 g) was run on a 2% agarose gel containing ethidium bromide for 1 h at 100 V and visualized under UV light. 2.7. Transmission electron microscopy L. infantum promastigotes, previously cultured for 4 days to late log phase of growth, were incubated in the absence or presence of 200 g/ml of KP or SP for 24 h at 25 °C and processed for transmission electron microscopy (TEM). The parasites were centrifuged (900g, 5 min), washed twice in PBS and Wxed in a solution containing 1% p-formaldehyde, 1.25% glutaraldehyde, and 0.5% sucrose in Sorënsen phosphate-buVer (0.92% NaH2PO4, 4.27% K2PO4, 0.1 M, and pH 7.2) for 2 h at room temperature. Afterwards, the cells were washed with 1.5% sucrose in the same buVer for 12 h, scraped oV and post-Wxed in a solution containing 2% OsO4 in Sorënsen buVer at 4 °C in the dark. The parasites were then dehydrated in ethanol, and embedded in a 1:1 mixture of Araldite M and Araldite Harter Hy 964 resins (Merck, Darmstadt, Germany) with an addition of 0.5% dibutyl-phthalate (Merck) and 2% accelerator DY 064 (Merck). Ultrathin sections at 70 nm (Ultracut UCT, Leica), picked up on 200 mesh copper grids and stained for 15 min with uranyl acetate and for 7 min in lead citrate (Merck), were washed, dried and then examined in a JEM1010 (JEOL, Tokyo, Japan) transmission electron microscope linked to a digital video-camera (Mega-View-III) with a software (Soft-Imaging-System, Münster, Germany) for the computerized acquisition of the images.

3.1. In vitro anti-leishmanial activity

3.2. Permeability assay KP treatment of L. major did not resulted in signiWcant permeability changes; methylene blue staining was shown in 5–17% of treated promastigotes, a value similar to control cells. Using a GFP-transfected L. major strain, membrane permeability was examined and the release of the Xuorescent protein in the culture supernatant (mean of two independent experiments in duplicate) was measured Xuorometrically. The results (Fig. 2) show that KP at concentrations greater than the LC50 did not cause leakage of intracellular GFP, whereas a large release of GFP into the supernatant was observed after amphotericin B treatment. 3.3. DNA fragmentation assay by agarose gel electrophoresis DNA analysis by agarose gel electrophoresis did not show any DNA fragmentation in L. infantum promastigotes treated with 100 or 200 g/ml of KP for 24 h (Fig. 3C), whereas DNA degradation was seen after miltefosine treatment (Fig. 3D). 3.4. Transmission electron microscopy To investigate which organelles are the targets of KP and the damages within the promastigote, ultrastructural analysis of L. infantum promastigotes treated with 200 g/ml of KP and SP for 24 h was performed in comparison with untreated control. Several alterations were observed in KP-treated leishmaniae (Fig. 5) compared with SP-treated (not reported) and untreated cultures (Fig. 4). The images of a normal promastigote (Fig. 4) show the presence of a normal Xagellum and Xagellar pocket (Fig. 4C), mitochondrion–kinetoplast

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Fig. 1. Growth curve of promastigotes of L. major (A) and L. infantum (B) in the absence and presence of Laminarinase (Lam) or KP at diVerent concentrations. 3,5

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B

C

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er ic

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Fig. 2. Cytoplasmatic retention of GFP molecules in KP (100 and 200 g/ml) and amphotericin B (0.1 and 0.2 g/ml)-treated promastigotes. The release of GFP was measured Xuorimetrically.

complex, nucleus, endoplasmic reticulum proWle, vacuoles, and the presence of glycosomes and acidocalcisomes; the same normal morphology was seen in SP-treated protozoa. In contrast, following KP treatment, severe structural damages and parasite ghosts were seen (Fig. 5). In particular, gross changes in the organization of the nuclear and kinetoplast chromatins and a widening of the nuclear membrane were detected (Figs. 5A and C). Moreover the mitochondrion–kinetoplast complex was intensely swollen (Figs. 5A and B), whereas the plasma-membrane appeared only

Fig. 3. Analysis by agarose gel electrophoresis. (A) Marker 1 kb DNA ladder; (B) L. infantum DNA (untreated control); (C) L. infantum after 24 h of KP (100 g/ml) treatment; (D) L. infantum after 24 h of miltefosine (25 M) treatment; (E) Marker 100 bp DNA ladder.

slightly ruZed with a normal layer of sub-pellicular microtubules (Fig. 5D); the Xagellum (Figs. 5B and D) and some glycosomes (Fig. 5B) also appeared normal.

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A

B F

F K G

A V

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Mi

K

N V

C

D

Mi

K K F

Fig. 4. Transmission electron microscopy of untreated cultures of L. infantum promastigotes. The protozoa have an elongated body and a normal aspect of the intracellular organelles. F, Xagellum; Mi, mitochondrion; K, kinetoplast complex; N, nucleus; V, vacuoles; G, glycosomes; A, acidocalcisomes. Bars: A–C, 1 m; D, 0.5 m.

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B F G K K F

G N

D

C

M K N

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Fig. 5. Ultrastructural alterations of L. infantum promastigotes after treatment with KP. The protozoa show an alterated ultrastructural morphology with cytoplasmic organelles disaggregated but the plasma-membrane appears to be intact. F, Xagellum; K, kinetoplast complex; N, nucleus; G, glycosomes; M, microtubules. Bars: A–C, 1 m; D, 0.5 m.

4. Discussion We have shown that KP, a killer decapeptide synthesized on the basis of a recombinant microbicidal anti-idiotypic antibody acting as a functional mimotope of a yeast KT (Polonelli et al., 2003), has in vitro leishmanicidal

activity against L. major and L. infantum, with an LC50 of 58 and 72 M, respectively. The activity was weak compared to conventional anti-leishmanial drugs, but KP demonstrated a lack of toxicity up to 500 g, as reported by Magliani et al. (2004). In the present study, we also report results obtained from the more virulent stationary

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phase (metacyclic) promastigotes, which are richer in lipophosphoglycans (Spath et al., 2000) and in the surface protease gp63 (Brittingham et al., 1995). Some results using log-phase leishmaniae show a greater activity of KP, as observed with a monoclonal antibody mimicking a yeast killer toxin (Savoia et al., 2002). In contrast to miltefosine, which causes an apoptotic cell death (Verma and Dey, 2004), KP treatment did not cause DNA degradation on L. infantum due to oligonucleosomal fragmentation. Our results are therefore consistent with cell death of leishmaniae after KP treatment via a non-apoptotic process. Membrane permeabilization has been shown to be important in protozoan killing by antimicrobial peptides, such as cathelicidins (McGwire et al., 2003) and temporins (Mangoni et al., 2005). These severely damage the parasite membrane causing dissipation of membrane potential and equilibration of intracellular pH with extracellular environment (Bera et al., 2003). Analogously, promastigotes exposed to miltefosine acquired rounded forms and an apoptotic cell death (Paris et al., 2004) occurred after amphotericin B treatment (Bera et al., 2003; Lee et al., 2002; Luque-Ortega et al., 2003), with a dissipation of ionic gradients or large lesions in the membrane. In contrast, KP treatment apparently did not aVect parasite membrane integrity, as demonstrated by the methylene blue exclusion assay, by the measurement of GFP Xuorescence release and by TEM analysis. Some earlier observations suggested that many host defense peptides exert their action by permeabilizing membranes to smaller molecules leading to energetic collapse of the promastigote in attempting to restore the lost ionic gradients (Bera et al., 2003; ZasloV, 2002). Our results also demonstrated that larger molecules such as GFP are retained within the cell; the electron microscopy data showed that in KP-treated promastigotes the plasma-membrane integrity appeared mostly unharmed and the microtubules were preserved. At the same time, the cytoplasm was disorganized and the main organelles to be aVected were the mitochondrion–kinetoplast complex structures with an evident swelling of the mitochondrion. The activity of KP on leishmania promastigotes may be governed by ionic and/or electrostatic interactions with the glycoconjugates present on the surface of the parasite inducing an autophagic cell degeneration and the abrogation of certain membrane functions. Previous observations with other KP-sensitive microorganisms, such as C. albicans (Polonelli et al., 2003), C. neoformans (Cenci et al., 2004), and P. brasiliensis (Travassos et al., 2004), suggested that the antimicrobial activity of KP was mediated by a still unknown mechanism based on its interaction with 1,3-glucan, the main KTR component (Guyard et al., 2002). Previously, we demonstrated a diVerent link to leishmania promastigotes through a monoclonal antibody mimicking a yeast killer toxin, which depended upon variation in expression of speciWc receptors on the cell surface of protozoa (Savoia et al., 2002). Overall, previous data and the demonstration here that laminarinase exerted a leishmanicidal

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activity similar to KP suggest the presence of 1,3-glucan on the cell surface of promastigotes. Hence the putative linkage to 1,3-glucan may be crucial for KP leishmanicidal activity. This is of great interest, since lipophosphoglycan and related glycoconjugates in leishmania can function as virulence determinants and appear to be ideal targets of chemotherapeutic intervention (Descoteaux and Turco, 1999; Mengeling and Turco, 1998; Turco et al., 2001). Furthermore, the leishmanicidal activity of killer monoclonal antibodies and of synthetic peptides interacting with 1,3-glucans suggests a possible role of these surface carbohydrate components in the induction of protective immunity. In the past, glucan was found to be a potent adjuvant when injected with diVerent leishmania antigens. Its adjuvant activity was supposed to be related to non-speciWc activation of macrophages (Holbrook et al., 1981; JareckiBlack et al., 1986; Obaid et al., 1989). Our Wndings suggest that 1,3-glucan may have a more speciWc role in eliciting protective anti-leishmanial immunity. Our observations oVer an interesting new approach for anti-leishmanial chemotherapy based on potentially broad antimicrobial spectrum molecules, such as KP, which may extend to many other pathogenic microorganisms possessing -glucan in their cell walls (Cenci et al., 2004; Magliani et al., 2004; Polonelli et al., 2003). Furthermore, as expected for peptides derived from physiological molecules such as antibodies, KP demonstrated the lack of any detectable toxicity to in vitro cultured cell lines as well as to white blood cells, as demonstrated by MTT colorimetric assays, supravital stains, and Xow cytometric methods (Magliani et al., 2004). Further studies are needed to assess the activity of KP against the intracellular amastigote stage of leishmania and its therapeutic (and/or adjuvant) activity in animal models of leishmaniasis. KP may represent the basis for modeling new and safe anti-leishmanial drugs with additional utility against other receptor-bearing microorganisms. References Bera, A., Singh, S., Nagaraj, R., Vaidya, T., 2003. Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides. Molecular and Biochemical Parasitology 127, 23–35. Brittingham, A., Morrison, C.J., McMaster, W.R., McGwire, B.S., Cang, K., Mosser, D.M., 1995. Role of the Leishmania surface protease gp63 in complement Wxation, cell adhesion, and resistance to complementmediated lysis. Journal of Immunology 155, 3102–3111. Cenci, E., Bistoni, F., Mencacci, A., Perito, S., Magliani, W., Conti, S., Polonelli, L., Vecchiarelli, A., 2004. A synthetic peptide as a novel anticryptococcal agent. Cellular Microbiology 6, 953–961. Conti, S., Fanti, F., Magliani, W., Gerloni, M., Bertolotti, D., Salati, A., Cassone, A., Polonelli, L., 1998. Mycobactericidal activity of human natural, monoclonal, and recombinant yeast killer toxin-like antibodies. Journal of Infectious Diseases 177, 807–811. Conti, S., Magliani, W., Arseni, S., Frazzi, R., Salati, A., Ravanetti, L., Polonelli, L., 2002. Inhibition by yeast killer toxin-like antibodies of oral Streptococci adhesion to tooth surfaces in an ex vivo model. Molecular Medicine 8, 313–317. Croft, S.L., Barrett, M.P., Urbina, J.A., 2005. Chemotherapy of trypanosomiases and leishmaniasis. Trends in Parasitology 21, 508–512.

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