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3
Natural Antimicrobial
Peptides from Eukaryotic Organisms
Renaud Condé, Martha Argüello,
Javier Izquierdo, Raúl Noguez, Miguel Moreno and Humberto Lanz
Centro de Investigación Sobre Enfermedades Infecciosas,
Instituto Nacional de Salud Pública
Mexico
1. Introduction
Antimicrobial peptides (AMP) are usually described as being short (less than 100 a.a.), gene
encoded, ribosome synthesized, polypeptide substances that have antimicrobial activity. For
simplicity reasons, we will exclude peptaibol and other non-ribosomaly synthetized
antibiotic from our classification.
The first peptidic antibiotic was described in 1968 coming from the Manduca sexta and was
of linear nature; since then the number of antimicrobial peptide discovered have grown
asymptotically. Though loose homology has been found between certain set of antimicrobial
peptides; it has proven difficult to classify the AMP through their primary structure.
Antimicrobial peptides show a great diversity of primary structures, and their short size do
not permit robust evolutionary classification, but for the most close related peptides. The
primary structures signature of the different AMP families may have arisen independently,
and in some case these structures homology are the result of convergent evolution rather
than a common ancestry. Nevertheless in order to classify the new components, general
classification methods have been established. So far this has been done regardless of
evolutionary relationship, source or activity. The criteria that have been commonly used are
the number of disulfide bridges and particular amino-acid composition. In 2005 P. Bullet
and co-workers suggested a 3 categories classification namely: α-Helical host defense
peptides (HDPs), β-Sheet HDPs, Flexible HDPs rich in certain amino acids (Bulet et al.,
1999). Though most AMP would fit in this classification, little insight about function can be
inferred from the class relation; nor does it give any comparative information between
peptides belonging to the same class.
More recently Tomas Ganz proposed a structural classification of the AMP based on their
secondary structure (Ganz, 2003b). The classes proposed included antimicrobial peptides
with 4 disulfide bridges with alpha helix and beta sheet mixed structures, 3 disulfide bridges
with alpha helix and beta sheet mixed structures, 3 disulfide bridges with beta sheet motif, 3
disulfide bridges with two alpha helix and beta sheet mixed structures, 2 disulfide bridges
with beta-sheet structures, one disulfide bridge cyclic peptide and alpha helical peptides.
The classification proposed here contains 9 different peptide structure families. The last group
consider hybrid structure peptide possessing structural features of more than one AMP class.
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Antimicrobial Agents
1.1 The lineal amphipathic alpha-helix antimicrobial peptide
1.1.1 General properties
The first peptide of this family discovered is the cecropin A from the pupae of the moth
Hyalophora cecropiae (Steiner et al., 1981; Hultmark et al., 1982). This structure of AMP has
been encountered in virtually all the multi-cellular organisms. Even if their sequences show
some similarity, they are not all evolutionary linked. As such, they cannot be aligned as a
whole, and are commonly separated in structural subclasses: Cecropin, magainin and
dermaseptin AMP. This AMP class do share general common features: the lack of cysteine
bridges, the tendency to form alpha helical secondary structure in relatively hydrophobic
solvent, the net positive charge at neutral pH and hydrophobic residues interspersed every
3 amino acid, giving them an amphipatic nature. Indeed, basic amino acid side chains face
predominantly one side of the alpha helix and hydrophobic residues are generally on the
other side of the molecule. A global alignment of the linear peptides separates three
different classes that could be broadly characterised as Dermaseptin, magainin and cecropin
class of lineal amphipathic AMP. Most of these peptides share the present a glycine near the
middle of their peptidic sequence.
1.1.2 Dermaseptins peptides
These peptides were extracted from Phyllomedusa genus frog skin secretions. Some of them
present a proline-induced kink in the middle of the alpha helix (Shin et al., 2001). Others
have a glycine in the same relative position that has been suggested to give the flexibility
needed for the membrane lysis activity (Xiao et al., 2006). This structural plasticity has been
defined as a molecular determinant for the antimicrobial vs eucaryont membrane specificity
(Shin et al., 1999; Shin et al., 2000; Shin et al., 2001), together with overall net positive charge
and hydrophibic moment. They present hydrophobic and positively charged amino acid in
an alternate pattern. Though mature dermaseptin amino acid sequences are highly variable,
their acidic pro-peptides are strikingly conserved (Azevedo Calderon et al., 2010).
1.1.3 Cecropin peptides
Cecropin peptides were first purified from insect hemolymph, and their expression is
usually inducible. Cecropins structural conformations were determined by NMR. Circular
dichroism and NMR (Nuclear Magnetic Resonance) data have shown that in aqueous
solvent the cecropin structure is largely disordered; but they adopt a stable alpha-helical
secondary structure in more hydrophobic environment. This makes the insertion of these
peptides in the lipidic membranes entropically favorable. Cecropin usually present a glycine
in the middle of their amino acid sequence. This glycine has been proposed to induce a kink
between the alpha helical structures that these peptides form in hydrophobic solvent. In
turn the deletion of this glycine or its replacement by another amino acid do not eliminate
the antimicrobial activity; instead it endows these mutant peptides with hemolytic and
cytolytic activity (Moore et al., 1996). The cecropin usually present a tryptophan in one of
the first two amino acid position as well as a glycine in the first position.
1.1.4 Magainin/scorpions AMP/cathelicidin peptides
Magainin peptides come from the frog genus Xenopus (Duclohier et al., 1989). In this AMP
class arthtropod AMP like Opistoporin-2 from Scorpion Opistophthalmus carinatus are also
Natural Antimicrobial Peptides from Eukaryotic Organisms
53
included (Moerman et al., 2002) as well as cathelicidin peptides (Travis et al., 2000) and the
fly cecropin from Stomoxys calcitrans (Boulanger et al., 2002). This phylogenetically
heterogeneous group present lysine/argine doublet repeats that could be considered as a
structural signature. These peptides do not present the conserved glycine present in the
other 2 lineal amphipatic AMP subclasses. Their positive general formal charge at neutral
pH is higher than the one of the cecropin and dermaseptin AMP. They also present a
conserved aspartic acid residue at the amino side of these peptides.
1.2 Proline rich peptides
This AMP class has been first described in mammals, in the intestine of Sus scrofa,
(Agerberth et al., 1991). They are also present in Hymenoptera, Lepidoptera and diptera.
Some of these peptides from this AMP class have been studied extensively, like drosocin
from D. melanogaster (Bulet et al., 1993), pyrrhocoricin from the European sap- sucking bug
Pyrrhocoris apterus (Cociancich et al., 1994) apidaecins from the Apis mellifera (Casteels-Josson
et al., 1993), and formaecin from the ant Myrmecia gulosa (Mackintosh et al., 1998). Mature
proline-rich antimicrobial peptides vary in length from 12 to 54 amino acid, and have few
common structural feature. Some authors distinguish between glycine/proline rich and
alanine/proline rich peptides. The variety of sequence does not allow for straightforward
sequence signature recognition for these peptides. Therefore, their antibacterial
activity/specificity is not deducible from the sequence analysis.
On the other hand, some of these peptides are able to penetrate the microbial cytoplasm
without inducing bacterial lysis, and do not present hemolytic or cytolitic activities
(Knappe et al., 2010). Model PR peptides have been designed, using the Ac-(Arg-Pro-ProPhe)n-NHCH3 framework, and some essential structural feature, necessary for
antimicrobial activity have been determined. The ability to form poly(proline)-II structure
in aqueous solution, as well as a critical peptide length are essential for antimicrobial
activity (Niidome et al., 1998).
1.3 Glycine/arginine rich peptides
The first purification of glycine rich peptide was done in 1991 (Bulet et al., 1991). As for the
proline rich antimicrobial peptide class, the glycine rich peptides have variable sizes and do
not show clear sequence signature, apart from the high proportion of glycine in their
primary sequence. These peptides are in general longer than AMP from other classes.
Between 25 to 50% of their amino acid are glycines. They have disordered structure in water,
and tend to self-order when in contact with artificial membranes (Bruston et al., 2007). The
structure of bombinin H resembles the influenza hemagglutinin fusion peptide (Zangger et
al., 2008). When binding to DPC micelles, a helix is formed that have a glycine ridge on one
side. There is an helix-helix interaction that leads to a multimerization process in the
bacterial membrane (Zangger et al., 2008).
1.4 Brevinin (hook structure) peptides
This class of peptides is characterized by having a short amino acid sequence, and a
carboxyloterminus disulfide bridge. Some brevinin peptides show post-translational
modification. The amino acids included in the carboxyloterminus loop are determinant for
the specificity of the antimicrobial activity as well as the length of the loop (Lee et al., 2002) .
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Antimicrobial Agents
The brevinin show alpha helical structure in sodium dodecyl sulfate solution (Lee et al.,
2002). Antibacterial activity is favored by structure that group the cationic amino acids of
the molecule with on one side an hydrophobic stretch of amino acids and on the other side
streches of apolar amino acids (Kumari and Nagaraj, 2001). Liposome disruption activity of
the brevinins correlates with the anti-Gram positive bacterial activity, suggesting a lytic
activity. None of the peptides showed hemolytic activity making brevinins attractive
prospects for broad-spectrum antimicrobial peptide design (Lee et al., 2002).
1.5 Defensins (cysteine knot structure)
1.5.1 General properties
This class of peptide is characterized by its rigid structure, given by the presence of 3 to 4
disulfid bridges. They are sub-classified through their cysteines connectivity and their
secondary structure.
By virtue of the cysteine knot motif that stabilize them, all these peptides show a rigid
secondary structure. Defensin are amphipatic molecules with a common defined beta sheet
motif secondary structure. Indeed there is a gamma-core motif (GXCX(3-9)C), considered
the structural signature of the disulfide-stabilized antimicrobial peptides that present two
beta strands with an interposed loop (Sagaram et al., 2011). This motif has one hydrophobic
and one hydrophilic side. The hydrophilic side of these peptides is usually constituted by
several lysine or arginine aminoacids. This gives them a general positive charge at
physiological pH. They are resistant to degradation and peptidase digestion because of their
compact structure. As for the other antimicrobial peptide classes, there are few phylogenetic
relationships even within each defensin subclass. The first three defensins class described
were found exclusively in mammals.
1.5.2 Alpha defensins
This type of defensins is found in mammals. Their cysteine are connected between the
cysteines 1-6 2-4 3-5. They show a structure of triple-stranded beta-sheet stabilized by a
conserved triple disulfide bridges array (Hadjicharalambous et al., 2008). Alpha defensin
sequence present more arginine than lysine residues, and it has been suggested that this
high arginine content endows the alpha-defensin with a higher antibacterial activity in high
salt conditions (Llenado et al., 2009).
P. hamadryas alpha-defensin ACYCRIPACFAGERRYGTCFYLGRVWAFCC
1.5.3 Beta defensins
The beta defensin have a cysteine connectivity of 1-5 2-4 3-6. They present the consensus
sequence of Xn-C-X2-4-G-X1-2-CX3-5CX9-10CX5-6CCXn (Ganz, 2003a) (C=cysteine and
G=Glycine). They present a tri dimensional structure of a triple stranded beta sheet. The
glycine invariant is also present in alpha defensin. This glycine is necessary for the beta
bulge structure to be formed, and the protein is unable to fold if it is replaced by any other
natural amino acid (Xie et al., 2005).
Sus scrofa beta-defensin 1 SVSCLRNKGVCMPGKCAPKMKQIGTCGMPQVKCCKRK
Natural Antimicrobial Peptides from Eukaryotic Organisms
55
1.5.4 θ defensins
θ defensin are macrocyclic octadeca peptides connected head to tail. These peptides are
present in monkeys but absent in human. There are θ-defensin ortholog pseudo genes in the
human genome but the Theta-defensin genes contain a premature stop codon that aborts
translation. (Cole et al., 2002) (Cole et al., 2004). Their synthesis implies a head to tail
circularization of an octa-peptide (Selsted, 2004). They were found to be active against S.
aureus, E. coli, and C. albicans as well as HIV virus (Cole et al., 2002). This type of defensins
also present a disulfide bridges stabilized amphipathic beta sheet structure.
P. hamadryas theta-defensin-1 RCVCRRGVCRCVCTRGFC
1.5.5 Insect defensins
The insect defensin class have an alpha-helix secondary structure bound to the beta sheet.
Their structures are similar to another arthropod peptide class: the scorpion potassium
channel blocker toxins (Bontems et al., 1991). Study of the structural features involved in the
antimicrobial activity of longicin, a defensin from the hard tick Haemaphysalis longicornis,
showed that the beta sheet alone was sufficient for antimicrobial activity. This part of the
insect peptide is positively charged at neutral pH, as does the alpha helix of the same,
nevertheless the role of the alpha-helix, at the antimicrobial level, seems to be restricted to
maintain the globular shape of the peptide (Rahman et al., 2009).
R. prolixus insect defensin B
ATCDLLSFRSKWVTPNHAGCAAHCLLRGNRGGHCKGTICHCRK
1.5.6 Plant defensins
Plant defensin antimicrobial peptide class present 4 disulfide bridges with a cysteine
connectivity of 1-8, 2-5, 3-6, and 4-7. Their three-dimensional structures are similar to the
insect defensin structure in that they have a disulfide bonds stabilized alpha-helix. Their
structure also show a triple anti-parallel beta-strands. (Sagaram et al., 2011). The strong
antifungal activity of the plant defensin has been associated with their alpha helix motif, in a
similar fashion as for insect defensin (Lamberty et al., 2001). Conversely, heliomycin, a
defensin from Heliothis virescens, has more structural communality with plant defensin than
with insect defensin (Lamberty et al., 2001).
P. sylvestris defensin 1
RMCKTPSGKFKGYCVNNTNCKNVCRTEGFPTGSCDFHVAGRKCYCYKPCP
1.6 Tachyplesin
This class of AMP was first found in horseshoe crab (Polyphemus litoralis). Gomesin is a
tachyplesin type of antimicrobial peptide found in tarantula hemocyte (Silva et al., 2000);
and androctonin has been extracted from scorpion hemolymph (Mandard et al., 1999). This
type of antimicrobial structure is broadly distributed amongst the genus. The AMP related
to this class of peptide present a beta sheet secondary structure stabilized by two disulfide
bridges (Nakamura et al., 1988). Tachyplesin have a rigid conformation of antiparallel betasheet connected by a beta-turn (Iwanaga et al., 1994). The tachyplesin family of AMP adopts
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Antimicrobial Agents
beta-hairpin-like structures when in contact with hydrophobic solvent. NMR studies
revealed a largely unordered structure in water, but a transition to a regular beta-hairpin
backbone conformation in the presence of dodecylphosphocholine micelles. The cysteine
null mutant of protegrin, a mammal tachyplesin type peptide, revealed that the cystein
bridges were not necessary for antimicrobial activity. Aside from their antimicrobial
activity, tachyplesin have also a scavenger capability. They bind lipo-polysacharide with
high affinity (Niwa et al., 1990). The structure of tachyplesin I also interacts with Vesicular
stomatitis virus envelope, inactivating the virus (Murakami et al., 1991).
Tachyplesin III Tachypleus gigas: KWCFRVCYRGICYRKCR
1.7 Tryptophan rich antimicrobial peptides
The archetypical W rich peptide is the indolicin, this peptide comes from Bos Taurus
neutrophils and is the result of proteolysis. Unlike the amphipathic alpha helical structure of
the cecropin class of peptides, their linear structure (no disulfid bridges) has no particular
secondary structure in water. Indolicin is globular and amphipathic in aqueous solution,
while it adopts a wedge shape when in contact with micelles. Indolicin shows a high
affinity for neutral POPC and anionic POPG vesicles(Hsu et al., 2005). The author suggests
that the structure changes and the strong membrane affinity are key to the antimicrobial
activity of indolicin (Ladokhin and White, 2001). Tryptophan rich AMP contains more than
25% of the aminoacid. Indolicin, the archetypical tryptophan rich antimicrobial peptide, has
a globular secondary structure in water, but show a wedge shape when in contact with in
lipid micelles (Rozek et al., 2000). This peptide has the ability to permeate bacterial
membranes and, depending of its tridimensional shape, inhibits DNA synthesis by binding
to it (Hsu et al., 2005).
Indolicin: H-ILPWKWPWWPWRR-NH2
1.8 Histidine rich glycoprotein peptides
Histidine-rich amphipathic cationic peptides are peptides with ¼ of their amino acids
represented by histidine. They show a global cationic amphipathic helical structure. They
trigger microorganism membrane disruption when the peptide adopts an alignment parallel
to the membrane surface. Even though, pore formation is not essential for their high
antimicrobial activity (Mason et al., 2009). Clavinin and daptomycin are other studied
members of this antimicrobial peptide class. Some Histine rich peptide, like LH4, also have
the capability to enhance transfection, a feature that is related to membrane perturbation
capability (Georgescu et al., 2010).
1.9 Mixed structure peptides
Some peptides share structural communality with more than one class of AMP; the sum of
the different AMP part activity are not additive; and these peptides classes do show unique
activity not present in the separated structural part of the molecule. Scorpine type AMP
represent a class on its own, as several homologous proteins have been found. The
structural defensin part of the molecule resembles the insect peptides. It has been
determined in later studies that once separated from the linear cecropin-like amino
Natural Antimicrobial Peptides from Eukaryotic Organisms
57
terminus, this peptide could effectively block these channels. Even though, the antimicrobial
activity of the complete molecule was dependent of the presence of this toxin/defensin
motif (Diego-Garcia et al., 2008).
The penaeidin class of peptide consist in proline-rich N-terminus and of a C-terminus
containing six cysteine residues engaged in three disulfide bridges (Destoumieux et al.,
2000). The proline-rich domain of penaeidin class AMP suffices to confer target specificity
and antimicrobial activity of penaeidin (Cuthbertson et al., 2004). The carboxyl end cysteinerich domain consists of an amphipathic helix linked to the upstream and the downstream
coils by two disulfide bonds. The peptide shows a highly hydrophobic core of globular and
compact structure, that has 2 arginines exposed on each side (Yang et al., 2003).
Another example of hybrid antimicrobial peptide is Hyastatin, isolated from the spider crab
(Hyas araneus) hemocytes (Cuthbertson et al., 2008). This AMP combines a Glycine rich motif
N-terminal region, a short Pro/Arg-rich region, and a panaeidin like C-terminal region
containing 3 disulfid bridges (Sperstad et al., 2009).
The chicken beta defensin 11 is formed by the repeat of two defensin motif, therefore having 6
disulfid bridges. This defensin show a nanomolar range of anti E.coli activity, being one of the
most effective antimicrobial peptide for this microorganism (Herve-Grepinet et al., 2010).
Microplusin, is a Rhipicephalus (Boophilus) microplus anti-microbial peptide (AMP).
Microplusin has a cysteine-rich AMPs structure with histidine-rich regions at the N- and Ctermini. Microplusin consists of five alpha-helix and has been shown to bind copper and
iron (Silva et al., 2009).
2. Antimicrobial mechanisms of AMP
The activity of AMPs must start at the cytoplasmic membrane since most AMPs permeabilize
microbial membranes. Several models have been proposed on how AMPs insert into the
membrane leading to the formation of ion channels, transmembrane pores or extensive
membrane rupture. These models are: 1) transmembrane pore models and 2) nonpore models
activity. Here we will also review other antimicrobial mechanisms that have been found. For
example, the antimicrobial mechanisms of apidaecin do not rely on pore forming activity, this
peptide does have antimicrobial activity at a concentration at least four order of magnitude
below the concentration that disturbs the bacterial membrane. Peptides from each structural
family have been reported to rely on antimicrobial mechanism that would not imply
membrane depolarization of the target microorganism, suggesting internal molecular
determinant. Certain peptides are unable to cause membrane depolarization at the minimal
inhibitory concentration, while other cause maximal depolarization well below the MIC
(Minimal Inhibitory Concentration) value. Evidences are mounting that involve particular
macromolecules as well as intracellular functions as the final target for antimicrobial activity
of the AMP. Even though, bacterial membranes are a necessary entry path for the AMP,
therefore determining part of the AMP selectivity as well as efficiency.
2.1 Transmembrane pore models of AMPs
There are more than 1,000 known AMPs (Brahmachary et al., 2004; Wang and Wang, 2004;
Fjell et al., 2007), and for the majority of them, there is little or no evidence for
58
Antimicrobial Agents
transmembrane pores (Wimley, 2010). Instead, there is compelling evidence that many
AMPs function by binding to membrane surfaces and disrupting the packing and
organization of the lipids in a nonspecific way. The simplest models of membrane
permeation by peptides involve the formation of membrane-spanning pores. These pores
have been studied in lipid bilayers and have been proposed to be the major cause of
bacterial membrane depolarization. As bacteria ATP synthesis is linked to the transmembranal potential of the cell, any perturbation of the ion partition may potentially lead to
the cell death. The structure of these peptides induced pores have been analyzed and several
pore structure were proposed.
The barrel stave pore model (Rapaport and Shai, 1991) involves a mechanism where the
peptides interact laterally with one another to form a specific structure enclosing a waterfilled channel, much like a protein ion channel. Bioinformatic analysis of protegrin 1
insertion in membranes concluded that this model was most consistant with the observed
energy of insertion of the peptide in artificial membranes (Langham et al., 2008).
Furthermore the electrophysiology record analysis of Ceratotoxin and pleuricidin peptides
inserted in lipid bilayers show that they form a peptide filled pore isolated from the lipids
from the membranes. The pore formation dynamics correlates with their antimicrobial
activity (Bessin, 2004).
In the toroidal pore model (Ludtke et al., 1996), specific peptide–peptide interactions are not
present Instead, the peptides affect cooperatively the local curvature of the membrane,
forming a peptide lipid toroid pore in the membrane. The cathelicidin peptide LL 37 (an
amphipathic, alpha-helical, antimicrobial peptide) appears to form such kind of pore in the
microbial membranes. NMR spectra studies of LL 37 shows that its pore channel is filled
with the membrane lipids phosphate heads. The resulting ionic leakage ultimately leads to
bacterial membrane depolarization (Henzler Wildman et al., 2003). The pore formation of
cateslytin, a beta sheet conformation peptide, has been studied through patch clamp and
NMR experiments, and has been found to fit a pore formation model involving transient
dissymmetry between the phospholipid leafs of the membrane as a key ingredient to
explain the formation of the pore (Jean-Francois et al., 2008)
The carpet model for AMP antimicrobial activity , originally described by Shai (Gazit et al.,
1996), is the most commonly cited model of membrane destabilization by AMPs. The
carpet/detergent model proposes that the accumulation of the peptides imbedded in the
microbial membrane provoques perturbation in the membrane integrity. Antimicrobial
peptides accumulate on the membrane surface with an orientation that is parallel to the
membrane untill peptide concentration has reached a critical level (i.e., a peptide-rich
‘‘carpet’’ has formed on the membrane surface). Then permeabilization occurs, via global
bilayer destabilization. PMAP-23, a cationic peptide member of the cathelicidin family, is
considered to induce membrane permeability according to the Shai-Matsuzaki-Huang
"carpet" model (Bocchinfuso et al., 2009). Cecropin P1, another alpha helical AMP,
imbedded in reconstituted phospholipid bilayer is preferentially oriented nearly parallel to
the surface of the lipid membranes, a position that is incompatible with the proteinaceous
pore model, as demonstrated by polarized ATR-FTIR spectroscopy analysis(Gazit et al.,
1996) The detergent model is also often cited to explain the catastrophic collapse of
membrane integrity, observed with some AMPs at high peptide concentration (Ostolaza et
al., 1993; Hristova et al., 1997; Bechinger and Lohner, 2006). Some authors combine the
Natural Antimicrobial Peptides from Eukaryotic Organisms
59
carpet and detergent models into a single idea in which the catastrophic collapse of
membrane integrity includes membrane fragmentation. Others distinguish between the two
models based on whether or not the peptide-induced leakage efficiency depends on the size
of the entrapped solutes (Ostolaza et al., 1993). Bechinger and Lohner (Bechinger and
Lohner, 2006; Salnikov et al., 2009) recently discussed molecular shape models in which
AMP lipid interactions could be depicted with phase diagrams to describe the propensity of
an AMP to permeabilize a membrane by disrupting the lipid packing. Epand and colleagues
have proposed a lipid clustering model in which AMPs induce clustering or phase
separation of lipids, with leakage occurring due to boundary defects (Epand et al., 2009;
Epand et al., 2010). Almeida and colleagues have described AMP activity in terms of
binding, insertion and perturbation using the sinking raft model, which they recently
augmented by adding a formal thermodynamic analysis to predict activity (Pokorny and
Almeida, 2004; Gregory et al., 2008; Almeida and Pokorny, 2009; Gregory et al., 2009;
Almeida and Pokorny, 2010). Interfacial activity is defined as the propensity of an
imperfectly amphipathic peptide to partition into the bilayer interface and drive the vertical
rearrangement of the lipid polar and non polar groups. The disruption of the normally strict
segregation of polar and nonpolar groups causes membrane permeabilization.
While these models, and others, have been useful in discussing AMPs, the molecular
organization of the lipid bilayer undergoing solutes leakage in the presence of AMPs is still
very much unknown. Without this knowledge, it remains challenging to predict structure–
sequence activity relationships; thus, it also remains challenging to engineer or de novo
design AMPs.
2.2 Non membrane mediated models of AMP activity
In addition to the pore models described above, AMP activity has been described using
antimicrobial mechanism that do not involve bacterial membrane permeability
impairement. For instance PR-39, a porcine proline-arginine-rich antibacterial peptide was
found to lyse the microbes through a non pore forming mechanism, altering the microbe
division and septum formation (Shi et al., 1996). Small Anionic antimicrobial peptides have
been found in ovine lungs, that appear to function without the need for the initial
electrostatic interaction with the bacterial membranes, and kill the bacteria through
intracellular content flocculation (Brogden et al., 1998). Mersacindin, a lantibiotic, has been
shown to inhibits bacterial cell wall formation (Brotz et al., 1997). Some antimicrobial
peptides like the pleurocidin and dermaseptin inhibit bacterial DNA synthesis while buforin
II and tachyplesin bind to nucleic acid in general (Yonezawa et al., 1992). The proline rich
AMP family target proteicious or nucleic acid intracellular molecules (Park et al., 2008),
indeed members of the proline-rich AMP family like Drosocin, pyrrhocoricin, and apidaecin
have been shown to act on bacterial GRO EL and Dna K proteins(Otvos et al., 2000).
3. AMP classical functions
The classical function of AMP has been their role as major effectors of the innate immune
system; AMPs complement the highly specific but relatively slow adaptive immune system.
Unlike the acquired immune mechanisms, endogenous AMPs, which are constitutively
expressed or induced, provide fast and effective means of defense. Most of these geneencoded peptides are mobilized shortly after microbial infection and act rapidly to
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Antimicrobial Agents
neutralize a broad range of microbes (bacteria, virus and protozoa). The ubiquitous nature
of antimicrobial peptides suggests that their role in nature has been long standing and must
have contributed to an organism’s fitness. Many of these molecules exert mechanisms of
action that appear to be unique and highly complex. However, AMPs exhibit varying, and
in some cases, significant degrees of host cytotoxicity, reflecting non-selective cell targeting
(Shin et al., 1999). It is likely that distinct antimicrobial peptides have evolved to function
within specific physiologic and anatomic contexts to minimize their potential to
concomitantly injuring the host cells. An intensive area of focus regarding antimicrobial
peptide biochemistry relates to the precise mechanisms by which these molecules cause cell
death. A long-held paradigm for microbicidal action has been that AMPs kill
microorganisms by initiating multiple injuries in target microbial cell membranes. The
principal theory suggest that peptides may create membrane pores in the organism, making
a leakage of some metabolites, ensuing depolarization, loss of membrane-coupled
respiration and biopolymer synthesis, and ultimately cell death. However, other authors
suggest additional mechanisms, , where membrane permeabilization alone appears to be
insufficient to cause cell death. These evidences come from studies documenting a clear
dissociation between membrane perturbation and cell death. In these cases, cell killing may
proceed in the absence of significant disruption in membrane architecture, due rather to
disruptions in cellular function (Zhang et al., 2000). The functional integrity of the
cytoplasmic membrane is crucial to essential functions of microbial pathogens, including
gradient formation and selective permeability, cellular energetics, and synthesis of biomolecules (Yeaman et al., 1998).
The general membrane effects of AMP are the membrane perturbation however alone may
be insufficient for microbicidal effects of certain peptides. Permeabilization alone does not
invariably result in staphylococcal death due antimicrobial peptides. Different peptides with
varying staphylocidal potencies exhibited disparate capacities of membrane
permeabilization and cell killing (Koo et al., 2001). Similar studies showed that gramicidin S
rapidly depolarizes Pseudomonas aeruginosa, but did not kill it, suggesting that the concept of
membrane perturbation and eventual cell killing may be independent (Zhang et al., 2000).
Bacterial membrane energetic also appears to be involved in AMP mechanisms of action
(Yeaman et al., 1998). It is now widely recognized that the AMP concept could play a
promising role in fighting the presently raging microbial resistance to conventional
antibiotics.
4. Unconventional function of AMP
4.1 Regulatory activities of AMPs.
Besides the role of endogenous antibiotics, the antimicrobial peptides have other functions
in the inflammation; wound healing and regulation of the response immune response, of
which are described below.
Microbial infection of the mucosa and skin induces production of large quantities of small
antimicrobial peptides, including defensins and cathelicidins, (Zasloff, 2002; Ganz, 2003a;
Yang et al., 2004). They can act as chemokines, such as some β-defensins chemoattract
immature iDC and other effector cells through the CCR6 receptor (Biragyn et al., 2002);
(Niyonsaba et al., 2004) or human cathelicidin LL-37 recruits neutrophils, monocytes and
mast cells via human formyl peptide receptor-like 1, FPRL1 (De et al., 2000) (Agerberth et
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61
al., 2000; Niyonsaba et al., 2002). Defensins can also activate effector cells that can work
together with the complement system to destroy microbial invaders. The α-defensins
HNP1~3 have been reported to increase the production of TNFa and IL-1 while decreasing
the production of IL-10 by monocytes (De et al., 2000). Some α-defensins enhance expression
of adhesion molecules including ICAM-1, CD11b, and CD11c by neutrophils and facilitate
the recruitment and enhance the microbicidal activity (Van Wetering et al., 1997; Chaly et
al., 2000; Di Nardo et al., 2003) (FÈger et al., 2002). β-defensins induce mast cell
degranulation and release of histamine and prostaglandin D2 (Yamashita and Saito, 1989;
Befus et al., 1999; Niyonsaba et al., 2001) increase the expression of CXCL8 and CXCL5 (Van
Wetering et al., 1997; van Wetering et al., 2002). Furthermore, murine β-defensin 2 has been
shown to act directly on immature DCs as an endogeneous ligand for Toll like receptor 4
(TLR-4), inducing up regulation of co-stimulatory molecules and DC maturation, triggering
robust, Th1 polarized adaptive immune responses in vivo (Biragyn et al., 2002). However,
the mechanisms that regulate these functions are not well studied. Defensins attract
inflammatory cells as neutrophils, B lymphocytes and macrophages. All these cells release
inflammatory mediators such as IL-8, IFNγ, IL-6, IL-10 and LTB4. It is interesting that
defensins may also have anti-inflammatory activity by the induction of IL-10 or SLPI.(Durr
and Peschel, 2002; Zasloff, 2002).The synthesis of β-defensins by epithelial cells and the
recruitment of peripheral blood granulocytes α-defensin-rich site of inflammation generates
a high concentration of them. Also have direct antimicrobial effects, defensins facilitate and
amplify the subsequent immune response. Indeed, spleen cells stimulated with α-defensins
increase the production of human cytokines and lymphocyte proliferation. This same type
of defensins, when administered to mice, produces increased serum IgG1, IgG2 and IgG2b.
In addition, small amounts of HNP extend the antibody response against a singenic tumor
(Tani et al., 2000).
These results indicate, without doubt, the AMPs have a role in the regulation of the
immune response. On the other hand, recent studies have identified several structurally
diverse endogenous mediators of innate immunity with certain features: firstly, they are
rapidly released in response to infection or tissue injury; secondly, they have both
chemotactic and activating effects on APCs; and thirdly, they exhibit particularly potent in
vivo immunoenhancing activity and enhance DC differentiation from DC precursors. This
subset of mediators alerts host defenses by augmenting innate and adaptive immune
responses to tissue injury and/or infection. On the basis of their unique activities, they are
called ‘alarmins’ (Oppenheim and Yang, 2005). Innate-immune mediators possessing
alarmin activity include defensins, cathelicidin, eosinophil-derived neurotoxin (EDN),
and high mobility group box protein 1 (HMGB1) (Oppenheim and Yang, 2005). The
concept of alarmins is very interesting. This has only been observed in mammals, but is
likely to exist in other groups of animals including insects. It has been observed overexpression of antimicrobial peptides during infection with various pathogens and even
damage to the cuticle. Many groups of insects have been used to understand the basic
characteristics of the innate immunity. However, surprisingly, the study of AMPs in
insects has been limited study of their bactericidal or antiparasitic activity and virtually
no information on the alternative role that could have the AMPs on the immune response
in these organisms.
Differential analyses after bacterial or fungal challenge showed the regulation of more than
a 100 molecules in adult Drosophila hemolymph (Levy et al., 2004). Using differential
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MALDI-TOF MS, 28 peptides with a molecular mass below 15 kDa and belonging to
different structural families were identified and could be classified into two groups. The first
group contains the AMPs and their different isoforms. DIMs belonging to this group are
likely to be effectors molecules of the immune response through their antimicrobial activity.
The second group contains molecules for which the lack of similarity to any peptide
prevents the proposition of any precise function. These peptides are suspected to serve as
chemokines during the Drosophila immune response but the different approaches for
investigating their role have so far been unsuccessful (Levy et al., 2004). On the other hand,
our group has analyzed the peptides in the hemolymph of mosquitoes An. albimanus
infected with malaria parasites. We found a complex pattern of peptides, including
cecropin, which are released into the hemolymph. Similarly, gambicin, cecropin, and
defensin are over-expressed in the intestinal epithelium and fat body of mosquitoes infected
with Plasmodium. However, it is unknown whether these peptides participate in the
elimination of the parasite. Cecropin has been consider an important AMP against
Plasmodium, but in vitro assays with synthetic cecropin did not affect Plasmodium viability
(unpublished results), but this peptide is over-expressed in mosquitoes infected with the
parasite (Herrera-Ortiz, A. et al., 2010.). It would be interesting to analyze the peptides
released into the hemolymph of these insects and their role in regulating the immune
response.
4.2 Anti-inflammatory (Anti-endotoxin) roles of host defense peptides
Bacterial lipopolysaccharides (LPS), also known as endotoxins, are major structural
components of the outer membrane of Gram-negative bacteria that serve as a barrier and
protective shield between them and their surrounding environment. LPS is considered to be
a major virulence factor as it strongly stimulates the secretion of pro-inflammatory cytokines
which mediate the host immune response and culminating in septic shock.
Early experiments determined that a number of host defense peptides from various sources
bound to LPS from diverse Gram-negative bacteria and reduced LPS-induced release of proinflammatory cytokines (e.g. TNF-α, IL-1, IL-6) and nitric oxide from monocyte or
macrophages and protected mice from LPS lethality (Larrick et al., 1994; Larrick et al., 1995;
VanderMeer et al., 1995; Kirikae et al., 1998). Initial studies focused on the unprocessed form
of cathelicidin, hCAP- 18 (Kirikae et al., 1998); however, it was later found that the LPSbinding properties of the peptide were contained within the processed 37-amino acid Cterminal domain, LL-37 (Turner et al., 1998). It has been proposed that the anti-endotoxic
properties of these peptides are the result of the inhibition of binding of LPS to CD14
(Nagaoka et al., 2001) and lipopolysaccharide binding protein (LBP) (Scott et al., 2000),
and/or indirect effects on cells (Scott et al., 2002). LL-37 has been shown to block a number
of LPS-induced inflammatory responses, including contractility and (nitric oxide) NO
release in aortic rings (Ciornei et al., 2003), pro-inflammatory cytokine production in a
macrophage cell line and in animal models (Scott et al., 2000; Ohgami et al., 2003),
suppression of leukocyte infiltration in a model of endotoxin-induced uveitis (Ohgami et al.,
2003) and lethality in animal models of sepsis (Scott et al., 2002). These effects occur at
concentrations in the physiological range for LL-37 (1–5 μg/ml) and may reflect a natural
role for LL-37 in the body (e.g. balancing of the potential stimulus by endotoxin from
commensals). This anti-endotoxin activity appears to correlate with an ability to dampen the
Natural Antimicrobial Peptides from Eukaryotic Organisms
63
pro-inflammatory effects of the Gram-positive surface molecule lipoteichoic acid (Scott et
al., 2002; Gutsmann et al., 2010) designed a new class of peptides synthetic anti-LPS peptides
(SALPs). SALPs were originally based on the LPS-binding domain of the Limulus anti-LPS
factor (LALF) but were substantially changed in length and primary sequence for optimal
binding to the lipid A portion of LPS. They observed that these peptides are highly efficient
in neutralization of LPS and blockage of its immunopathological consequences in vitro and
in vivo. SALPs combine excellent selectivity for LPS, with high neutralizing activity in vitro
and potent protection to septic shock using the murine model in vivo. They also demonstrate
the biological efficacy of rationally designed new synthetic antilipopolysaccharide peptides
(SALPs) based on the Limulus anti-LPS factor for systemic application. Efficient inhibition of
LPS-induced cytokine release and protection from lethal septic shock in vivo was analyzed,
whereas cytotoxicity was not observed under physiologically relevant conditions and
concentrations. It seems that the lipid A part of LPS is converted from its “endotoxic
conformation,” the cubic aggregate structure, into an inactive multilamellar structure. These
observations suggest a novel therapeutic role of AMPs.
4.3 Anti-viral activity
Apart from the antibacterial activity, AMPs also possess antiviral activity. For example, the
α-defensins target the human immunodeficiency virus (HIV) activity by directly
inactivating viral particles and affecting the ability of the virus to replicate within CD4 cells.
Human α-defensins HNP-1 to -3 and HD-5 have been shown to block papillomavirus
infection. Retrocyclin 2, a synthetic θ-defensin peptide that humans do not synthesize due to
a mutation in the corresponding human gene, has the capacity to block influenza virus
infection. Human β-defensins can also block HIV-1 replication, and interestingly, a single
nucleotide polymorphism in a β-defensin gene has been associated with clinical
manifestation of HIV-1 infection, suggesting that the human β-defensins play an important
role in host defense against HIV. Cathelicidins, in contrast, have an inhibitory effect on
lentiviral replication in vitro, and LL-37 appears capable of interfering with vaccinia virus
replication in vitro and in mice. Dermaseptin S4, a 28-residue AMP isolated from frog skin,
attenuates HIV infection in vitro. Other AMPs from frog skin including caerin 1.1, caerin 1.9,
and maculatin 1.1 have also demonstrated inhibition of HIV in vitro(Albiol Matanic and
Castilla, 2004).(Daher et al., 1986; Sinha et al., 2003; Yasin et al., 2004). Our group has worked
with the peptide named scorpine from the venom of Pandinus imperator scorpion, where we
observed a very interesting anti-virus dengue and anti-plasmodium activity (CarballarLejarazu et al., 2008). Scorpine is an antimicrobial peptide whose structure resembles a hybrid
between a defensin and a cecropin. It exhibits antibacterial activity and inhibits the sporogonic
development of parasites responsible for murine malaria. The recombinant expressed scorpine
(RScp) in Anopheles gambie cells showed antibacterial activity against Bacillus subtilis and
Klebsiella pneumoniae, at 5 and 10 µM, respectively. It also produced 98%mortality in sexual
stages of Plasmodium berghei at 15 µM and 100% reduction in Plasmodium falciparum parasitemia
at 5 µM. RScp also inhibited virus dengue-2 replication in C6/36 mosquito cells. In addition,
we generated viable and fertile transgenic Drosophila that over-expresses and correctly secretes
RScp into the insect hemolymph, suggesting that the generation of transgenic mosquitoes
resistant to different pathogens may be viable. However, there is no knowledge of their
mechanics, action. It is necessary to extend these studies with other peptides during infection
induced with virus dengue and other pathogens.
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5. Evolutionary perspective on antimicrobial peptides
In this chapter, we proposed a structural classification of antimicrobial peptide families
considering the diversity of their structures and then we reviewed the traditional function
and biological activities. Finally, we propose new insights into the functions of antimicrobial
peptides that could provide a large body of research to create new classes of antimicrobial
therapeutics. AMPs are widespread molecules throughout the animal and plant taxa, this
fact suggest its relevance in the evolution of immune response. Traditionally, their basic
molecular and biochemical nature is related to the disruption of membrane potential and/or
structure with the ensuing cell death. However, the diversity in the structure and biological
properties (above mentioned) of AMPs within and between species suggest that these
molecules have different functions in immune response.
The immune system of living organisms is formed by a set of cells, molecules and reactions.
All of these features are continuously evolving to resist (attack and eliminate) pathogen
invasion and to limit the negative (in terms of host survival and reproduction, i.e. fitness)
consequences of the infection (Hoffmann and Reichhart, 2002). On the other hand,
pathogens success depends upon overcoming the selective immune pressures brought about
by the host. As a consequence, both, host and pathogens, evolve traits and strategies to
increase the fitness of each one. Van Valen (Van Valen, 1973) proposed this co-evolutionary
arm races as an evolutionary theory called “The Red Queen Hypothesis”. The theory was
proposed citing Lewis Carrol’s Red Queen, where it takes all the running somebody can do
to keep in the same place. Given this situation, immunologically, there is never a “best”
solution to pathogens infection. To understand this scenario, we must consider (1)
pathogen’s short generational cycles, that may provide enough time to adapt to the host’s
immune response. As an outcome there will be grounds for high variability in immune
response. (2) Differences in the kind and burden of pathogens, where divergent hostpathogen interactions for each species are possible and can be reflected in the course of
action of taxa immune response (Read and Taylor, 2001). (3) Virulence differences, where
there can or cannot be a harm imposed on a host (for example Bacillus anthracis vs.
commensal microbiota). As long Hypothesis, an immune effecter that has the ability to be
produced under different infection circumstances could have a selective advantage,
antimicrobial peptides could have this property, as the host has to deal with the problem of
maximizing fitness under the Red Queen pressure.
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