Thus, when injected into anopheline mosquitoes previously infected with a variety of Plasmodium spe-cies, the antimicrobial peptides disrupted sporogonic Keywords antimicrobial peptide;
Trang 1Multifunctional 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.
Trang 2development 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-blockinduc-ing 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 IC50: 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.
Antiparasitic effect (l M ) Reference
Tetrahymena pyriformis MIC = 8.1 Acanthamoeba castellani MIC = 0.9
Leishmania pifanoi LC50= 14.4
SSGHCSPSLKCWCEGC
a Acyl-lysyl oligomers where: l, dodecanoyl; L, aminododecanoyl; K, lysyl Underlined letter designates amino acids in the D -configuration.
Trang 3ookinete and gamete developmental stages of
Plasmo-dium berghei with ED50 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
Trang 4tachylep-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 stephensimosquitoes [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 (IC50 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 amprom-astigotes (LC50 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 (IC50 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 (IC50 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 K4K20-S4 or its short analog K4S4(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 K4S4(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
Trang 5aminoheptanoylated 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 (IC50 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 LC50
(hemolysis) to IC50 (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
Trang 6Fig 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-K4-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].
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.
Trang 7manner 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 (IC50
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 Nonetheless, these studies strongly
sug-gest that the physicochemical attributes resulting from
the molecular structure of antimicrobial peptides can
be useful in engineering selective and efficient
antipara-sitic therapeutic drugs
Acknowledgement
This research was supported by the Israel Science Foundation (grant 283⁄ 08)
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