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MINIREVIEW Multifunctional host defense peptides: antiparasitic activities Amram Mor Department of Biotechnology & Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel Introduction Human parasites are responsible for millions of deaths around the world every year. Malaria, for instance, is endemic in over 100 Third World countries, with an estimated 400 million clinical cases correlating with 1–3 million deaths annually, and over 3 billion inhabit- ants of tropical regions are considered to be at risk [1]. Of particular concern is the causative agent of human malaria, Plasmodium falciparum, a large number of strains of which are drug resistant, in particular to chloroquine [2]. Reports of field strains of P. falcipa- rum demonstrating in vitro resistance to artemisinins – recently introduced antimalarial drugs – are also alarming [3]. Similarly, leishmaniases are important causes of morbidity and mortality in humans and ani- mals on four continents, and are extremely difficult to treat [4]. There is thus a clear need for new therapeutic agents against these and related parasites. A multitude of preliminary studies suggest that host defense pep- tides (HDPs) represent a promising route towards developing new, efficient antiparasitic therapies [5]. Magainins and cecropins were among the very first examples of animal HDPs reported to display antipar- asitic activities, some 20 years ago [6], and synthetic hybrids of cecropin and melittin have exhibited enhanced potency [7]. These notorious antimicrobial peptides are known to function as part of an inducible immune response against a number of microbial infec- tions. Thus, when injected into anopheline mosquitoes previously infected with a variety of Plasmodium spe- cies, the antimicrobial peptides disrupted sporogonic Keywords antimicrobial peptide; chemical mimicry; drug design; drug resistance; infectious disease; Leishmaniasis; malaria; membrane active compound; oligo-acyl-lysyl; peptide-mimetic Correspondence A. Mor, Laboratory of Antimicrobial Peptides Investigation (LAPI), Department of Biotechnology & Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel Fax: +972 4 829 3399 Tel: +972 4 829 3340 E-mail: amor@tx.technion.ac.il (Received 28 April 2009, revised 12 August 2009, accepted 12 August 2009) doi:10.1111/j.1742-4658.2009.07358.x Whereas significant knowledge is accumulating on the antibacterial and antifungal properties of host defense peptides (HDPs) and their synthetic mimics, much less is known of their activities against parasites. A variety of in vitro and in vivo antiparasitic assays suggest that these notorious anti- microbial compounds could represent a powerful tool for the development of novel drugs to fight parasites in the vertebrate host or to complement current therapeutic strategies, albeit the fact that HDPs essentially act by nonspecific mechanisms casts serious doubt on their ability to exert suffi- cient selectivity to be considered ideal candidates for drug development. This minireview summarizes recent efforts to assess the antiparasitic prop- erties of HDPs and their synthetic derivatives, focusing on two of the most used models – Plasmodium and Leishmania species – for antiparasitic assays against the different development stages. Abbreviations APP, antilipopolysaccharide factor; HDP, host defense peptide; Hst5, histatin-5; OAK, oligo-acyl-lysyl. 6474 FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS development by aborting the normal development of oocysts. As the vector cannot transmit the parasite to another host, this suggested the possibility of induc- ing effective transmission-blocking immunity in the mosquito vector by transgeneic expression of genes coding for magainins, cecropins or similarly acting parasiticidal peptides in the mosquito genome [6]. Since then, antiparasitic activities have been reported for numerous additional antimicrobial peptides and their synthetic derivatives. Representative antiparasitic peptides (APPs) are listed in Table 1, and reports from the past decade are briefly outlined below. APPs Among the structurally constrained peptides, defensins are by far the most investigated family of HDPs, and numerous members have been reported to exert a vari- ety of antiparasitic activities. For instance, the classic antifungal defensin, drosomycin, was shown to signifi- cantly inhibit the development of Plasmodium berghei ookinetes at micromolar concentrations [8]. Scorpine, a 75-mer peptide from scorpion venom Pandinus impe- rator whose structure resembles a hybrid between defensin and cecropin, was reported to inhibit both the Table 1. List of representative natural and synthetic antiparasitic peptides. MIC and IC 50 : minimal concentrations inducing 100% and 50% inhibition of parasite growth, respectively. MLC and LC 50 : minimal concentrations inducing 100% and 50% lysis of the parasite, respectively. Compound Primary structure Parasite Antiparasitic effect (l M) Reference Magainin II GIGKFLHSAKKFGKAFVGEIMNS Paramecium caudatum MIC = 4.1 36 Tetrahymena pyriformis MIC = 8.1 Acanthamoeba castellani MIC = 0.9 Cecropin–melittin KWKLFKKIEKVGQGIGAVLKVLTTGL P. falciparum IC 50  10 7 D-HALO-rev AKKLOHALHOALLALOHLAHOLLAKK P. falciparum IC 50 = 0.1 24 Hst5 DSHAKRHHGYKRKFHEKHHSHRGY Leishmania donovani LC 50 = 7.3 26 Leishmania pifanoi LC 50 = 14.4 NK-2 KILRGVCKKIMRTFLRRISKDILTGKK T. cruzi IC 50 = 2.5 15 P. falciparum LC 50 = 6.2 14 Angiotensin II NRVYIHPF P. gallinaceum IC 50  30 16 aI-Defensin ACYCRIPACIAGERRYGTCIYQGRLWAFCC T. cruzi IC 50  937 hII-Defensin Cyclic GVCRCLCRRGVCRCLCRR Leishmania amazonensis IC 50  12.5 11 Drosomycin DCLSGRYKGPCAVWDNETCRRVCKEEGR SSGHCSPSLKCWCEGC P. berghei IC 50  10 8 Gomesin ZCRRLCYKQRCVTYCRGR P. falciparum IC 50  80 22 P. berghei IC 50  50 Buforin II TRSSRAGLQFPVGRVHRLLRK C. parvum IC 50  20 20 Bombinin H4 I IGPVLGLVGSALGGLLKKI L. donovani IC 50 = 1.7 17 L. pifanoi IC 50 = 5.6 Temporin A FLPLIGRVLSGIL L. donovani LC 50 = 8.4 18 L. pifanoi LD 50  12 Tachyplesin KWCFRVCYRGICYRRC L. braziliensis LD 50 = 4.7 21 T. cruzi LD 50 = 87.8 Tyrothricin A Cyclic VOLFPFFNQY P. falciparum IC 50 = 0.0006 27 Gramicidin S Cyclic VOLFPVOLFP P. falciparum IC 50 = 1.3 27 K 4 K 20 S4 ALWKTLLKKVLKAAAKAALKAVLVGANA P. falciparum IC 50 = 0.2 30 K 4 S4(1–13) ALWKTLLKKVLKA P. falciparum IC 50 =6 31 L. major MLC = 12.5 28 C 3 -K 4 S4(1–13) Propanoyl-ALWKTLLKKVLKA P. falciparum IC 50 =4 31 L. major MLC = 18 28 NC 7 -K 4 S4(1–13) Aminooctyl-ALWKTLLKKVLKA L. major MLC = 6.25 28 P. falciparum IC 50  532 Dermaseptin O1 GLWSTIKQKGKEAAIAAAKAAGQAALGAL T. cruzi MLC = 2.8 38 L. amazonensis MLC = 23 39 OAKs a lK-LK-LK-LK P. falciparum IC 50 = 0.08 35 LK-LK-LK P. falciparum IC 50 = 0.14 a Acyl-lysyl oligomers where: l, dodecanoyl; L, aminododecanoyl; K, lysyl. Underlined letter designates amino acids in the D-configuration. A. Mor Antiparasitic peptides FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS 6475 ookinete and gamete developmental stages of Plasmo- dium berghei with ED 50 values of 0.7 and 10 lm, respectively [9]. There are newer reports of transgenic scorpine producing 98% mortality in the sexual stages of P. berghei at 15 lm and a 100% reduction in P. falciparum parasitemia at 5 lm [10]. Various defen- sins also have leishmanicidal properties, possibly by inducing apoptosis [11]. The fact that substantially higher leishmanicidal activity was observed against mutant strains of Leishmania major in which leishman- olysin, a surface metalloprotease, was knocked out suggests that virulence factors such as leishmanolysin might prevent antimicrobial peptide-mediated apop- totic killing. Longicin, another member of the defensin family, from the tick Haemaphysalis longicornis, exhib- ited antiparasitic activity in vitro and in vivo against the erythrocyte stage of Babesia, the merozoites, by preventing or retarding their proliferation. The fact that longicin exerted a hemoparasiticidal effect without demonstrable toxicity against mammalian host cells suggests its usefulness as a model for the development of chemotherapeutic compounds against tick-borne disease organisms [12]. A recent report studied clinical isolates from three microsporidia species, using spore germination and enterocyte-like cell infection assays to determine the effect of a panel of antimicrobial peptides: lactoferrin, lysozyme, human b-defensin-2, human a-defensin-5, and human a-defensin-1. The antimicrobial peptides have been reported to efficiently inhibit spore germination and ⁄ or cell infection of one or a number of isolates, either alone or upon combina- tion with lysozyme, suggesting that intestinal anti- microbial peptides contribute to the prevention of infection by luminal microsporidia spores [13]. The linear 27-residue synthetic derivative of the porcine protein NK-lysin, NK-2, was found to exert selective activity against P. falciparum [14]. Infected human erythrocytes were rapidly permeabilized by NK2 at 5–10 lm, which reduced the viability of the intracellular parasite, whereas noninfected cells were hardly affected. This selectivity was ascribed to loss of plasma membrane asymmetry and concomitant exposure of phosphatidylserine upon infection. NK-2 was also reported to kill the intracellular parasite Try- panosoma cruzi, the causative agent of Chagas disease, leaving the host cell unharmed [15]. Angiotensin II and a related peptide, vanicere-5, were studied in culture and in mosquitos for their effects on the development of Plasmodium gallinaceum sporozoites. When injected into the insect thoraxes, these peptides reduced infection intensities in the mos- quito salivary glands by 88% and 76%, respectively. Although the mechanism of action is not fully under- stood, the authors proposed that these peptides selec- tively disrupt the cell membrane of P. gallinaceum, and showed additionally that preincubation of sporozoites in vitro with vanicere-5 reduced the infectivity of the parasites with regard to their vertebrate host [16]. Bombinin H4 is a native antimicrobial peptide of animal origin with a single l-amino acid to d-amino acid isomerization in position 2, which improves its biostability. Bombinin H4 was reported to affect the viability of both insect and mammalian forms of Leish- mania by rapidly perturbing their plasma membranes at micromolar concentrations. The mode of action involved rapid (within minutes) depolarization and loss of integrity of the plasma membrane, associated with rapid bioenergetic collapse [17]. The 13-residue-long temporins show leishmanicidal activity while maintaining biological functions in serum. Their lethal mechanism is believed to involve plasma membrane disruption, on the basis of the observations that the peptides induce rapid collapse of the plasma membrane potential, influx of exogenic molecules, and reduced intracellular ATP levels [18]. Buforin activity against Cryptosporidium parvum was strongly dependent on the parasite life cycle: the oocyst was barely affected after 3 h of incubation with 10 lm buforin, whereas the sporozoite’s viability decreased by almost 100%. The authors speculate that the sporozoites are susceptible because their cytoplas- mic membrane is somewhat structurally similar to the bacterial cytoplasmic membrane [19]. This group also showed that the moderate activity of buforin can be enhanced upon combination with azithromycin or minocycline, > 90% parasite reduction being observed at the highest concentration tested [20]. A recent study investigated the antiparasitic activi- ties of different antimicrobial peptides isolated from aquatic animals. These included penaeidin-3 from the shrimp Litopenaeus vannamei , the antilipopolysaccha- ride factor (ALF) from Penaeus monodon, clavanin A from the ascidean Styela clava, an analog of magainin (MSI-94) from the frog Xenopus laevis, tachyplesin I from the limulid Tachypleus tridentatus, and mytilin A from the mussel Mytilus edulis. These antimicrobial peptides were selected because of their previously dem- onstrated potent effects against bacteria, yeasts, and filamentous fungi. The antiparasitic activity was eval- uated against the promastigote form of Leishmania braziliensis as well as against the epimastigote and trypomastigote forms of Trypanosoma cruzi. Tachylep- sin was found to be the most potent peptide in killing both L. braziliensis and T. cruzi, and was therefore suggested to be the most suitable candidate for further investigation as a therapeutic agent [21]. A tachylep- Antiparasitic peptides A. Mor 6476 FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS sin-related peptide called gomesin was tested against asexual, sexual and presporogonic forms of P. falcipa- rum and P. berghei parasites. When added to culture of P. berghei mature gametocytes, gomesin inhibited the exflagellation of male gametes and the formation of ookinetes. In vivo, the peptide reduced the number of oocysts of both Plasmodium species in Anophe- les stephensi mosquitoes [22]. Attacin is an immune effector peptide that has been shown to inhibit the growth of Gram-negative bacte- ria. In Glossina morsitans morsitans, which serves as the sole vector of African trypanosomes, attacin was implicated in trypanosome resistance and in maintain- ing parasite numbers at homeostatic levels in infected individuals [23]. Studies of the histidine-rich antimicrobial peptide LAH4 resulted in several active derivatives [24]. The most selective APP was a 26 amino acid analog, D-HALO-rev, which showed high potency (IC 50 of 0.1 lm) against the human malarial parasite P. falci- parum, and concentrations toxic against erythrocytes and fibroblasts at least two orders of magnitude higher than those needed for its antiplasmodial activ- ity. The mechanism of the antiplasmodial activity is unclear; however, important differences in the mem- brane composition of Plasmodium spp. versus the host cells are predicted to enhance the activity of designed antiplasmodial peptides (for a recent review see [25]). By contrast, another histidine-rich APP, human histatin-5 (Hst5), seems to target mitochon- drial ATP synthesis [26]. In the human parasitic pro- tozoan Leishmania, mitochondrial ATP production is crucial, as the organism lacks the bioenergetic switch between glycolysis and oxidative phosphorylation described in some yeasts. Hst5 displays activity against both stages of the parasite life cycle, prom- astigotes and amastigotes (LC 50 values of 7.3 lm and 14.4 lm, respectively). Hst5 was proposed to induce a lethal effect by causing limited and temporary dam- age to the plasma membrane of the parasites, as assessed by electron microscopy, membrane depolar- ization, and uncontrolled entrance of a vital dye. Fol- lowing this initial interaction, Hst5 translocates into the cytoplasm of Leishmania in a nonstereospecific, receptor-independent manner, accumulates in the mitochondrion, and produces bioenergetic collapse of the parasite by decreasing the synthesis of mitochon- drial ATP. Tyrothricin, a complex mixture of antibiotic peptides produced by Bacillus brevis, was reported in 1944 to have antimalarial activity rivaling that of quinine in chickens infected with P. gallinaceum. More than 60 years later, the major components of tyrothricin, cyclic decapeptides collectively known as the tyroci- dines, were isolated and tested against P. falciparum. Although the tyrocidines differ from each other by conservative amino acid substitutions in only three positions, their parasite inhibitory concentrations spanned three orders of magnitude (IC 50 of the most potent compounds ranged between 0.6 and 360 nm). For comparison, gramicidin S, a structurally analogous antibiotic peptide tested under the same conditions, was not as active (IC 50 of 1.3 lm) but exerted anti- parasitic activity by rapid lysis of infected erythro- cytes. Like those of previously described antimicrobial peptides, tyrocidine activities correlated strictly with increased apparent hydrophobicity and reduced total side chain surface area due to the presence of ornithine and phenylalanine in key positions. Unlike antiplasmo- dial activity, however, mammalian cell toxicities of the respective peptides were considerably less variable, ranging only from 2.6 to 28 lm [27]. Various native members of the frog-derived derm- aseptin family exhibit potent antiparasitic properties against both Leishmania [28] and P. falciparum [29]. Synthetic derivatives of dermaseptin S4, such as the 28-mer K 4 K 20 -S4 or its short analog K 4 S4(1–13), dis- played enhanced activity towards human erythrocytes infected with P. falciparum, killing the parasite through lysis of the host cells [30]. Both derivatives were more efficient in inhibiting parasite growth at the mature trophozoite stage than at the younger ring stage. This fact supports the view that the antiplasmodial effect is essentially derived from lysing the host cell membrane; that is, because the host cell membrane evolves with parasite maturation, trophozoites are expected to be more sensitive than rings, as observed [30]. Various conjugation derivatives of K 4 S4(1–13) were assayed with the aim of avoiding lysis of host cells. These derivatives have established that increased hydropho- bicity at the N-terminus invariably results in an ampli- fied antiplasmodial effect, irrespective of the linearity or bulkiness of the additive. However, increased hydrophobicity was also generally associated with increased hemolysis and lack of discrimination between infected and noninfected erythrocytes [31]. By contrast, aminoacyl counterparts were generally more selective [32]. Thus, as compared with the parent peptide, the aminoheptanoylated version displayed both increased antiparasitic efficiency and reduced hemolysis, includ- ing against infected cells. Presumably, by selectively dissipating the parasite plasma membrane potential and causing depletion of intraparasite potassium, this derivative exerted more than 50% growth inhibition at peptide concentrations that did not cause detectable hemolysis. Hence, unlike the parent peptide, the A. Mor Antiparasitic peptides FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS 6477 aminoheptanoylated derivative was not stage-selective, being equally inhibitory for both the ring and tropho- zoite stages. Additional new members of the derma- septin family also act as APPs, displaying potent activity against Leshmania (e.g. dermaseptins O1 and H3) and against T. cruzi (e.g. dermaseptins D11 and D12) [25,38,39]. Chemical mimicry of APPs In addition to the naturally occurring APPs and their de novo designed synthetic derivatives, recent studies suggest that potent antiparasitic properties can be generated from HDP-mimetic compounds designed to mimic the structure and ⁄ or function of the native peptides [33,34]. The potential therapeutic use of anti- parasitic HDPs is likely to be significantly limited by several major obstacles pertaining to less than ideal properties, including relative toxicity and bioavail- ability issues as well as a relatively high production cost. By reproducing the critical biophysical character- istics of HDPs, peptidomimetics might better address these issues while endowing resistance to degradation enzymes. Recently developed HDP mimetics, termed oligo- acyl-lysyls (OAKs), were based on a linear sequence of alternating aminoacyl chains and cationic amino acids so as to mimic the prototypical sequence of linear HDPs. Like potent HDPs, OAKs can display rapid, nonhemolytic, broad-spectrum microbicidal properties both in vitro and in vivo. Various OAK sequences were shown to inhibit the growth of different plasmodial strains (IC 50 range 0.08–0.14 lm). Further investiga- tions performed with a representative OAK revealed that the ring and trophozoite stages of the parasite developmental cycle were equally sensitive to this com- pound, unlike the case with the parent dermaseptin peptides. A shortcoming of the OAK was the need for long incubation times in order for it to exert its full effect [35]. Nevertheless, certain OAKs displayed highly selective antiparasitic activity, the ratio of LC 50 (hemolysis) to IC 50 (parasite growth inhibition) being > 10 000 for the most selective OAK, composed of only three acyl-lysyl subunits (Table 1). These results indicated that the OAK did not exert its antimalarial action by lysis of infected erythrocytes, as was the case with the parent dermaseptins, and pointed to the potential of the OAK system to generate simple, highly selective and low-cost compounds that might be useful in fighting malaria. Note that, although the OAKs rep- resent, so far, the only HDP mimetic system able to generate antiparasitic compounds, it is anticipated that future studies of various existing and ⁄ or new future mimic strategies will reveal interesting antiparasitic properties. Because of their chemical robustness, such compounds are likely to overcome various drawbacks of conventional antimicrobial peptides, including sus- ceptibility to proteases, and might therefore be both useful investigation tools and new, promising candi- dates for therapeutic developments. Possible mechanisms for APPs Attempts to understand the mechanism(s) underlying the observed antiplasmodial activity suggest that the activities of distinct APPs obey many of the rules governing their ability to disrupt bacterial membranes (discussed extensively elsewhere in this issue). This type of interaction appears to be acutely influenced by the respective charges and amphipathies of the reactants. In fact, a comparison between the peptides’ ability to inhibit the growth of malaria parasites and that of bacteria demonstrates a remarkable parallelism in the way that each modification affects both activities, as assessed with numerous substitution ⁄ truncation deriv- atives [28–31]. Additional support for this view is provided by the results obtained with experiments on kinetics and the dissipation of the membrane potential, as well as from the fact that activity is rapid and is independent of a chiral center [32]. Therefore, a variety of experimental evidence suggests that the mode of antiplasmodial activity of some antimicrobial peptides might be based on selective membrane disruption, despite the fact that the parasite’s membrane is well hidden within its host cell. As shown in Fig. 1, it is speculated that, owing to differences in membrane composition [25,40], APPs have a higher affinity for the membranes of infected erythrocytes than for those of normal erythrocytes (hence the often observed greater extent of hemolysis of infected cells), but must have a still higher affinity for the parasite’s membrane. The observed labeling of intracellular parasites in non- lysed, infected erythrocytes (i.e. under nonhemolytic conditions) supports this view (Fig. 1), although the apparent differential distribution of rhodaminated peptides may also be due to an experimental artefact: the fluorescence may be collisionally quenched by hemoglobin, which is present only in the host cell com- partment. Nevertheless, colocalization evidence exists (although unpublished) suggesting that, in macro- phage-infected amastigotes, the antileishmanial activity also might proceed by direct interaction of dermasep- tin with the intracellular parasite (Fig. 2). Thus, when dermaseptin, for instance, binds the membrane of a hosting erythrocyte, the peptide is somehow able to transfer to the parasitic membrane in an affinity-driven Antiparasitic peptides A. Mor 6478 FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS Fig. 1. Proposed model for APP interaction with infected erythrocytes. The left panel shows phase-contrast and fluorescence confocal microscope images providing evidence for the direct interaction of the rhodamine-labeled dermaseptin derivative, aminohexyl-K 4 -S4(1–13), with unfixed intraerythrocyte P. falciparum trophozoites. The right panel is a cartoon illustration of two hypothetical modes of action. The upper drawing shows the initial adhesion of the lipophilic APP (blue) to the erythrocyte membrane. Subsequently, hemolytic APPs can assemble to disrupt that membrane (right drawing). The resulting hemoglobin leakage will lead to parasite starvation. Nonhemolytic APPs (left drawing) can undergo an affinity-driven transfer (through lateral diffusion?) from the erythrocyte to the parasite membranes. The lower drawings represent potential APP fates once they have reached the parasite’s membrane: (1) superficial carpet-like interactions can modify the membrane properties (e.g. charge and fluidity of lipids and protein components) and interfere with their proper normal functions [40]; (2) APP internalization and interruption of vital biological processes (e.g. DNA functions) [34]; (3) APPs can also disrupt the parasite’s plasma membrane (similarly to the hemolytic process) as evidenced by the APP’s ability to dissipate the parasite plasma membrane potential and cause depletion of intraparasite potassium under nonhemolytic conditions [32]. A B C D Fig. 2. Proposed model for APP interactions with infected macrophages. The left panel shows the experimental colocalization of APP and parasites in a human macrophage infected with L. major amastigotes, as visualized by fluorescence confocal microscopy: (A) rhodaminated dermaseptin applied to unfixed cells; (B) fluoresceinated antiparasite antibody applied after cell fixation; (C) merged image of (A) and (B); (D) phase-contrast image of the infected macrophage shown in (A), (B) and (C). The right panel is a cartoon illustration of two hypothetical routes leading to the colocalization of dermaseptin and parasite in an infected macrophage. In the upper drawings, the cartoon shows that cationic APPs could reach the cytoplasm by diffusion, exploiting the negative-inside transmembrane potential or via vesicle-like internalization followed by fusion with the parasitophorous vacuole. The subsequent events that might follow are described in the lower drawings of Fig. 1. A. Mor Antiparasitic peptides FEBS Journal 276 (2009) 6474–6482 ª 2009 The Author Journal compilation ª 2009 FEBS 6479 manner and to exert a membrane-lytic activity upon the pathogen. Namely, such a ‘transfer’ could physi- cally occur through the new permeability pathways that are induced by the parasite in the membrane of the host erythrocyte. As the parasite is completely engulfed within a parasitophorous vacuole membrane, solutes which leave or enter the parasite must therefore traverse three membranes: that of the host erythrocyte, the parasitophorous vacuole membrane, and the para- site membrane. Experimental evidence for the ability of APPs to gain access to the parasite in malaria- infected cells, obtained using confocal microscopy analysis of labeled dermaseptins, is shown in Fig. 1 [32]. The data showed that, in infected cells, the labeled peptide crossed the erythrocyte plasma mem- brane and concentrated in internal compartments, although it is presently unclear what was the peptide’s ultimate target, e.g. the parasite membrane, the mito- chondrion, or nucleic acids. Antimicrobial peptides are known to target each and ⁄ or all of the above [34]. Conclusions Although the mechanism of action of most antimicro- bial peptides is far from being fully understood, the vast majority are now believed to act by one or even a combination of different nonspecific mechanisms that can target not only the cell membrane integrity but also extracellular and intracellular processes (reviewed by Pierre Nicolas in [41]; this issue). Such a multitarget mode of action is in good agreement with the observed large spectrum of sensitive microorganisms, and signifi- cantly reduces the likelihood of emergence of efficient resistance mechanisms. Thus, even though the antipar- asitic properties have not been investigated thoroughly, as yet, an increasing number of convincing studies seem to support the view that the antiparasitic activity of antimicrobial peptides also emanates from interac- tions with multiple targets. Most remarkably, however, at least a few peptides exhibit very high potency (IC 50 values in the nanomolar range) and a selectivity factor of several orders of magnitude. These selective com- pounds appear to be endowed with the formidable ability to cross a number of membrane systems before specifically disrupting a target(s) in the intracellular parasite. Although differences in membrane composi- tion are likely to contribute to this selectivity, the molecular basis for these observations remains largely ill-understood. 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MINIREVIEW Multifunctional host defense peptides: antiparasitic activities Amram Mor Department of Biotechnology & Food Engineering,. 289–294. 41 Nicolas P (2009) Multifunctional host defense peptides: Intracellular-targeting antimicrobial peptides. FEBS J 276, doi:10.1111/j.1742-4658.2009.07359.x. Antiparasitic peptides A. Mor 6482. and antifungal properties of host defense peptides (HDPs) and their synthetic mimics, much less is known of their activities against parasites. A variety of in vitro and in vivo antiparasitic assays