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Science against microbial pathogens: communicating current research and technological advances

______________________________________________________________________________
A. Mndez-Vilas (Ed.)

Antibacterial Peptides: A Review


Christine Czard, Viviane Silva-Pires, Catherine Mulli and Pascal Sonnet
Laboratoire des Glucides, quipe THERA, CNRS UMR 6219, UFR de Pharmacie, Universit de Picardie Jules Verne,
1 Rue des Louvels, 80037 Amiens, France

Antibacterial peptides (APs), small molecules composed of less than fifty amino acid residues, are important components
of the innate immune system. APs are produced by almost all living organisms in a defense strategy against invading
pathogens. APs can be divided into many subtypes following different criteria: origin, size, structure, mechanism of action,
etc... Despite their great diversity, APs share properties such as their ability to fold into amphipathic conformations, their
affinity for phospholipids, their amphiphilic nature, APs have been shown to kill bacteria rapidly by acting on disrupting
the bacterial membrane in a non-specific way. Depending on the nature of the AP, many different modes of action have
been proposed. Nowadays, APs are a potential replacement for antibiotics, as resistance among pathogenic bacteria is
increasing and has become a global and growing public health problem. Throughout this review, we will focus on natural
and synthesized cationic antibacterial peptides, which are likely to show antibiotics properties. We will present and discuss
their secondary structure, mechanism of action, potential targets and role as therapeutic agents.

Keywords antibacterial peptide; mechanism of action; pathogenic bacteria

1. Introduction
In 1928, Alexander Fleming discovered Penicillin, the first natural antibiotic. In the mid-30s, Prontosil, the first
commercial antibiotic, became available. Following World War II, drug companies conducted searches to find new
antibiotics and up to the 60s, many new classes were discovered: antibiotic chemotherapy was then in its golden age.
Their (over)use was wide spread as they greatly improved the quality of human life treating a wide variety of illnesses
caused by bacteria. Starting from the 80s, a slowdown was observed: on the one hand very few antibiotic chemical
structures were discovered but improvements within existing classes were still proposed and on the other hand, a
resistance to nearly all antibiotics in clinical use was observed. These past forty years, only three new classes of
antibiotics, targeting Gram negative bacteria only, entered the market. Today, multi-antibiotic(s) resistance among
pathogenic bacteria is evolving and expanding to the point of becoming a global and growing public health problem.
Finding alternatives to traditional antibiotic chemotherapy is hence urging and was recently highlighted by the
European Centre for Disease Prevention and Control (ECDC)/ European Medicines Agency (EMEA) in their joint
technical report and interviews: A future without effective antibiotics will exacerbate a situation where already at least
25,000 patients in the EU each year die from infections due to multidrug-resistant bacteria. Patients suffering from
healthcare-associated infections will be particularly hard hit.[1]
At the same time of early antibiotics observations, antimicrobial activities were discovered in secretions of human
origin. Since then, numerous antimicrobial substances were isolated from a wide range of organisms, some of which
show a selectivity towards both Gram negative and Gram positive bacteria. Antimicrobial peptides are important
components of the innate immune system and are produced by nearly every living organism in a defense strategy
against invading pathogens. Consequently, the ability of these natural compounds to interact with bacteria has raised
interest for promising pharmacological and therapeutic applications. Indeed, compared to antibiotics, these peptides kill
bacteria rapidly, have broader activity spectra and furthermore, they are not affected by resistance mechanisms such as
those witnessed for antibiotics. Several thousands of such peptides have been identified, as nearly each species of living
organism produces its own kind of antibacterial peptides. Nowadays, as few new antibiotics are proposed, the short size
and efficient action of antibacterial peptides (APs) added to their lack of specificity towards a target makes them very
promising drug candidates, at the condition of overcoming severe limitations. Indeed, their in vivo activity is decreased
compared to what is generally observed in vitro; at higher concentration APs can be cytotoxic to mammalian cells and,
last but not least, the cost of production to develop potent antibacterial peptides is huge.
Throughout this review, we will give an overview of antibacterial peptides, their origin, their biological functions, their
modes of action, their potential resistance to bacteria and their future as therapeutic agents as an alternative to
antibiotics.

2. What is an Antibacterial Peptide?


Antibacterial peptides (APs) are small molecules generally composed of fewer than fifty amino acid residues mostly in
their common L configuration. APs can be divided into many subtypes following different criteria: origin, size,
structure, amino acid sequence, biological action, mechanism of action, etc... but it has been shown that secondary
structure is the only meaning criterion to sort them.[2, 3] Hence, four classes of APs have been proposed: -sheet, -

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helical, loop and extended peptides (see Fig. 1).[4] However, many APs possess two domains, -sheet and -helical for
instance,[5] while some others cannot be classified. The vast majority of APs have a net positive charge (i.e. cationic
peptides, rich in basic amino acids such as arginine and/or lysine), but a minor subgroup composed of anionic peptides
which will not be discussed here, does exist.[6-8] Despite their great diversity, APs share properties such as their ability
to fold into amphipathic conformations, their affinity for negatively charged phospholipids, their amphiphilic nature,
Indeed, cationic APs contain hydrophilic sequences on one side aligned with hydrophobic sequences on the opposite
side. APs, which are synthesized by nearly all living organisms (plants, animals, humans, micro-organisms, ) are
encoded by the genome. They are produced through regular processes of gene transcription and ribosomal translation.

(a) (b)

(c) (d)

Fig. 1 3D model structures taken from the RCSB Protein Data Bank (http://www.pdb.org/), representing the differences between
the four classes of cationic peptides. (a) -helical peptide, corresponding to the structure of magainin 2 (PDB ID: 2MAG[9]). (b) -
defensin 1 is a peptide composed of a series of -sheets (PBD ID: 1KJ5[10]). (c) The loop structure of the gramicidin peptide (PDB
ID: 1MAG[11]). (d) An example of an extended peptide, corresponding to an indolicidin analogue (PDB ID: 1QXQ[12]).

2.1 Cationic linear -helical antibacterial peptides


The -helical peptides family is the largest, the most common in nature and the most studied class of cationic peptides.
They have been identified in plants, vertebrates and invertebrates. This subgroup contains approximately 250 linear
peptides with antibacterial activity, generally composed of less than forty amino acid residues other than cysteine.[13]
They present a notable amphipathic behavior, are highly positively charged and possess a tertiary structure with a kink
or a hinge in the middle.[14, 15] These peptides are unstructured in aqueous solution, and fold into their -helical
configuration upon binding the bacterial membrane where they are either absorbed onto its surface or inserted into it. A
direct correlation has been established between -helical conformation and antibacterial activity.[16]

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2.2 Cationic -sheet antibacterial peptides


This subgroup is composed of approximately 250 -sheet peptides that are conformationally constrained and stabilized
because of the presence of one to five disulfide bridges.[13] They thus adopt a more or less cyclic conformation. They
already exist in their -sheet conformation in aqueous solution and may be further stabilized upon binding the bacteria
membrane. The number of disulfide bridges has an impact on the overall structure as well as on the activity of the
peptide. It has been shown that the cyclic structure was essential for antibacterial activity.[17, 18]

2.3 Cationic linear extended antibacterial peptides


These peptides present an unusual amino acid composition. They are linear in shape and are characterized by an
overexpression of one or more amino acid. This subgroup contains nearly 90 peptides that do not show any secondary
structure either in -helix or in -sheet. Some of them are rich in histidine residues, like histatin[19, 20] found in human
saliva, whereas other such as PR-39 are rich in proline and arginine residues or as prophenin, rich in proline and
phenylalanine. These peptides are very flexible in solution.

2.4 Cationic loop antibacterial peptides


Proline-arginine rich peptides cannot form amphipathic structures because of the overexpression of proline residues.
Instead they adopt a polyproline helical type-II structure.[21]
It has to be noted that any living organism can produce different classes of the aforementioned APs, including a number
of variant of a same given class. According to Hancock et al.[22] there are at least four possible reasons for this
structural diversity among APs. First, although the activity spectrum of any given AP is quite broad, it will not be active
against every pathogen the host might encounter. Thus, a diversity of APs with distinct but yet overlapping activities
increases the hosts innate defense system vs. pathogens. Second, APs showing different structures may work together
in a better synergy. Third, the non-antibacterial features of these APs, such as chemotactic or pro-inflammatory
activities, also vary from one class to another. Finally, different cells types produce different types of APs, thus
complementing each other.
As of today, more than a thousand natural APs have been isolated and characterized from different sources. These APs,
along with several thousands of synthetic variants, have been gathered in numerous on-line databases, which are in
constant expansion. Mid-April 2011, the Antimicrobial Peptide Database[23] (http://aps.unmc.edu/AP/main.php)
contained 1747 antimicrobial peptides, 1372 of which have an antibacterial activity. Of the latter, 268 are active against
Gram positive bacteria only and 129 against Gram negative only. 42 APs present a negative net charge while 63 are
neutral. Table 1 presents the repartition of these 1372 APs among the different classes.

Table 1 Repartition of the 1372 antibacterial peptides among the different structural classes obtained from the
Antimicrobial Database (last accessed on April 18th, 2011).

Secondary Structure Number of APs % of total


-helix 253 19
-sheet 63 5
Mix of -helix and -sheet 27 2
Extended 87 6
Disulfide bridge(s) 182 13
Unknown 760 55

3. Mechanisms of action
The mode of action of a vast majority of APs to kill bacteria is based on membrane disruption followed by pore
formation on the nanometer scale and membrane depolarization. The following general model for the mechanism of
action has been proposed: (i) AP-membrane attraction, (ii) attachment of the AP onto the membrane and (iii) insertion
of the AP into the membrane causing its disruption, leading to the leakage of ions and metabolites. -helical peptides
have been the most studied so far, whereas in comparison little is known in comparison about the mechanism of action
by which -sheet peptides to permeabilize the membrane.

Attraction:
Numerous studies demonstrated that the net charge of APs was directly correlated with their attraction/interaction with
the bacterial membrane. Indeed, cationic APs possess a positive net charge, ranging from +2 to +9 at physiological pH,
while bacteria present a highly negatively charged outer membrane due to the presence of phosphate groups within
lipopolysaccharides (LPS) for Gram negative bacteria and within lipoteichoic acids for Gram positive bacteria,

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respectively.[24] Attractive electrostatic interactions between APs and the outer membrane of the bacteria then occur
bringing the two moieties together. It has been demonstrated that, up to a certain threshold value, the more positively
charged the AP, the better the antibacterial activity and selectivity.[25]

Attachment insertion :
APs are now in close vicinity to the bacterial surface. The initial electrostatic interactions lead to an actual and
nonspecific association of the AP with the bacterial membrane. In the case of Gram negative bacteria, cationic APs will
bind the outer membrane via the anionic phosphate groups of the LPS. Gram positive bacteria neither have an outer
membrane nor LPS moieties at their surface for the AP to bind to. Instead, their surface presents peptidoglycan
featuring anionic teichoic acid groups, on which the APs will be able to attach. It has to be acknowledged that it is
because of the negative charge borne by the membrane that APs are able to differentiate bacteria from a host cell, thus
preventing APs from being toxic. The structure of APs is such that, opposed to their cationic side, there is a
hydrophobic side. APs can aggregate, forming cluster deposited onto the membrane. In vitro studies showed that
depending on several parameters such as the nature of the AP, that of the membrane or the peptide concentration, APs
bind to the bacterial surface in two different states, namely an S state and an I state,[26] where S stands for Surface
and I for Insertion. Basically, at a low peptide-to-lipid ratio, the peptides tend to be adsorbed onto the surface, adopting
an orientation parallel to the bilayers. They stay in a functionally inactive S state inducing a thinning/stretching of the
membrane. As the peptide-to-lipid ratio increases and reaches a threshold depending mostly on the lipid composition of
the bilayer, the orientation of APs changes and becomes perpendicular to the bilayers, i.e. APs switch to the I state
and begin the process of insertion into the membrane, eventually resulting in pore formation. After binding the bacterial
membrane, APs will go through conformational changes to adopt energetically favorable secondary structures dictated
by their hydrophobicity. For example, -helical peptides will adopt orientations that are parallel or perpendicular to the
membrane.[27] Moreover, membrane permeation is a concerted process involving clusters of APs, as it is energetically
unfavorable for an -helical peptide to pass through the membrane as a monomer.
Depending on the nature of the AP, many modes of action based on this model have been proposed: barrel-stave
model, carpet model, toroidal pore model, these models differing mainly in the attachment-insertion step. The in vivo
mechanisms and the precise description of the AP-membrane interactions are still controversial and it is important to
point out that membrane disruption is a complex phenomenon involving a combination of complex mechanisms.
Besides, some cases where the peptide does not act on the membrane to form pores but rather accumulates inside the
bacteria cell or affects intracellular functions have been reported as well and are presented in section 3.4.

3.1 Permeabilization mechanism 1: The barrel-stave model


In 1977 Ehrenstein et al.[28] proposed the first mechanism to explain bacteria killing by APs. APs accumulate as
monomers on the bacterial surface, but forming circle patterns. Upon binding, APs adopt an orientation parallel to the
lipid bilayer. They then reorient perpendicularly and insert into the lipid core of the membrane resulting in a shape
resembling a barrel whose staves are the -helical APs.[29] During this process, APs undergo conformational phase
transition: the hydrophobic surfaces of the APs face outward, towards the acyl chains of the membrane thus aligning
with the lipid core of the bilayer, while the hydrophilic regions face each other and form the interior of the pore.
Progressively, new APs are recruited and through a process of self-aggregation, the pore size increases as more and
more APs adopt a trans-membrane configuration. In this model, the membrane is neither deformed nor bent during the
insertion process. Indeed, the AP inserts itself in the bilayer by drilling the membrane.

3.2 Permeabilization mechanism 2: The carpet model


In 1992, Pouny et al. studied dermaseptin S (a cationic amphipathic -helical peptide isolated from frog skin) and
demonstrated that the interaction of this AP with membranes clearly diverged from the barrel-stave model. A new
mechanism, namely the carpet model was then proposed.[30] Like for the barrel-stave model, APs aggregate onto the
bilayer surface but keep a parallel alignment to the membrane surface during the process.[31] The peptides are bound to
the bacterial surface on their hydrophobic side while their hydrophilic side faces the exterior. Clusters of APs eventually
coat the bacterial surface in a carpet-like way. As the concentration of APs increases, the membrane is weakened by
unfavourable energetics and APs become likely to intercalate into it in a detergent-like fashion causing the membrane to
break up into micelles and further dissolve. This mechanism does not involve pore formation and APs do not insert into
the membrane. For this mode of action to be efficient, the concentration of APs must be high as they must cover the
whole bacterial membrane. It has to be noted that in contrast to the barrel-stave and the toroidal pore model, APs do not
need to adopt a specific structure (e.g. -helical) to permeabilize the membrane.

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3.3 Permeabilization mechanism 3: The toroidal pore model (a.k.a. The wormhole mechanism)
This third model, proposed by Hancock et al. in 1999,[32] combines the actions of the barrel-stave and carpet models.
The APs aggregate on the membrane similarly to the carpet model and then insert themselves perpendicularly into it,
causing its deformation.[33] Unlike in the barrel-stave model, lipids are intercalated with APs in the transmembrane
channel in formation. Upon interacting with the membrane, APs adopt an -helical structure, and orient themselves
parallel to the membrane surface enabling the binding to occur between the polar face of the APs and the polar head
group of the lipids, causing the membrane to bend.[34] Its curvature is such that the pore is lined by both the APs and
the lipid head groups, whereas the lipid tails interact with the hydrophobic surface of the APs.[29] Eventually, toroidal
pores will be formed in the membrane, leading to its disruption. The difference with the two previous models is that
during the insertion process, the APs remain permanently bound to the LPS moieties of the membrane.

3.4 Intracellular targets


Many studies indicate that membrane disruption is often not sufficient to lead to bacteria death. In some cases, the
killing of bacteria may occur with very little to no membrane disruption. Some evidence suggests that there are
intracellular targets as well.[35] Some APs, like Buforin II, a proline -helical AP, do not permeabilize the bacterial
outer membrane, but penetrate it to accumulate in the cytoplasm, exerting its cytotoxic activity.[16] The mechanism of
translocation involves a concerted action with other APs. APs rich in arginine are able to translocate across both the
cellular and nuclear membranes, where they interact with DNA, RNA and/or proteins to inhibit synthesis pathways.[36]
There is no general scheme to describe these mechanisms as they are specific to one AP. Once translocated in the
cytoplasm APs are able to act on many levels: inhibition of cell-wall synthesis, inhibition of enzymatic activity,
inhibition of DNA, RNA and protein synthesis, binding to DNA, altering cytoplasmic membrane (inhibition of septum
formation) and/or activation of autolysin.

4. Principal families of cationic antibacterial peptides and their activity spectrum


Amongst the multi-resistant drug bacteria responsible for nosocomial infections are Staphyloccocus aureus,
Pseudomonas aeruginosa, Escherichia coli, In this paragraph, we will describe the principal families of cationic
APs active against these pathogens (see Table 2). Most of APs present a broad activity spectrum and show activity
against the aforementioned bacteria. The most active APs present a minimum inhibitory concentration (MIC) of 1 to 4
g.ml-1, corresponding to the minimum concentration of APs that completely prevents bacterial growth (see Table
3).[37]

Table 2 Examples of natural APs along with their secondary structure, origin and sequence (adapted from references
[24] and [38]). Subscript pairs identify amino acids that are joined by disulfide bonds and * means amidated at the C-
terminus.

Peptide Structure Source(s) Sequence


Magainin 2 -helix frog GIGKFLHSAKKFGKAFVGEIMNS
SMAP-29 -helix sheep RGLRRLGRKIAHGVKKYGPTVLRIIRIAG
LL-37 -helix human LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES
HNP1 -sheet human AC1YC2RIPAC3IAGERRYGTC3IYQGRLWAFC2C1
hBD1 -sheet human DHYNC1VSSGGQC2LYSAC3PIFTKIQGTC2YRGKAKC1C3K
PR-39 Extended pig RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFP*
Histatin Extended human RERPPIRRPPIRPPFYPPFRPPIRPPIFPPIRPPFRPPLRFP
Indolicidin Extended bovine ILPWKWPWWPWRR*
Gramicidin A Loop bacteria VGALAVVVWLWLWLW
Bactenecin Loop bovine RLC1RIVVIRVC1R

4.1 Magainins
Magainins 1 and 2, also known as peptide glycine serine (PGS) were first isolated in the late 80s from the skin of the
African frog Xenopus laevis.[39, 40] More generally, magainins are found within amphibian skins, as well as in
mammals. The name magainin derives from the Hebrew magain meaning shield. Magainins 1-2 are composed of 23
amino acids, exhibit a net charge of +4 and differ by two residues in position 10 and 22. They adopt an amphipathic -
helical structure and present a broad antimicrobial activity spectrum (they are, among others, active against both Gram
positive and Gram negative bacteria, see Table 3). Their average calculated MIC is comprised between 50 and 150
g.ml-1 for standard strains of bacteria. Analogs showing an enhanced activity have been synthesized.[39] Their mode
of action follows the toroidal pore model to permeabilize the bacterial membrane.[41]

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4.2 Cathelicidins
Cathelicidins are cationic bi-partite peptides constituted by a highly conserved N-terminal domain of approximately 100
residues, named cathedin and by a C-terminal region. They have been isolated in neutrophilic granules and epithelial
cells of many invertebrate and vertebrate species, including reptiles, fishes, birds, mammals and even humans.[42-47]
The C-terminal domains of cathelicidins are of varied length (from 12 to 100 amino acids) and structures, hence
cathelicidins share little homology beyond the cathedin domain. By their chemical structure, cathelicidins are a very
diverse group of molecules. Among the most studied cathelicidins are SMAP-29 and LL-37. SMAP-29 has been
isolated in sheep and is, as of today, one of the most potent AP known, its MIC against some bacteria being below 1
g.ml-1. It is composed of 29 residues, adopts an almost all- helical conformation and presents an amphipathic
nature.[48, 49] Its antibacterial activity is attributed to its N-terminal amphipathic -helix.
LL-37, the only cathelicidin found in humans, adopts an -helical conformation, bears a net charge of +7 and is
constituted of 37 amino acids. LL-37 disrupts the bacterial membrane though the toroidal pore model[50] or through the
carpet model.[51]

4.3 Defensins
Together with cathelicidins, defensins constitute the two principal AP families present in mammals[52] and are the best
characterized. They were first discovered in 1983 in rabbits[53] and two years later in humans.[54] Following these
discoveries, numerous defensins were isolated from other living organisms as well (plants, invertebrates, micro-
organisms, etc) Defensins are cationic peptides, rich in cysteine residues, adopting mainly -sheet structures.
Mammalian defensins contain six cysteine residues and are classified into three subclasses differing by the alignment of
the three stabilizing disulfide bridges.
-defensins: Around 50 kinds of -defensins have been discovered, six of which of human origin. Their length
varies from 29 to 35 amino acids and they adopt a triple-stranded -sheet conformation. The disulfide bridges are
located between residues Cys1-Cys6, Cys2-Cys4 and Cys3-Cys5. Human neutrophils express four distinct -defensins,
referred to as Human Neutrophil Peptides-1 to 4 (HNP1-4).[55] The remaining two human -defensins, labeled Human
Defensin 5 and 6 (HD5-6), are found in Paneth cells of the small intestine and in epithelial cells.[56, 57]
-defensins: 90 types of -defensins have been isolated, constituted of 36 to 50 amino acids. The disulfide
linkages are between residues Cys1-Cys5, Cys2-Cys4 and Cys3-Cys6 and like -defensins, they adopt a triple-stranded -
sheet conformation. Nearly 30 -defensins have been identified in humans by genome analyses (gene-based
searches),[58] but only six have actually been found. Their length is of 41 to 50 amino acids and they are labeled
Human Beta Defensin x (HBDx), with x ranging from 1 to 6. HBD1-6 are expressed by epithelial cells.[59]
-defensins: So far, only three have been found in monkeys,[60] and it has been shown that they have been
inactivated in humans.[61] In contrast to - and -defensins, -defensins adopt a total circular conformation through the
Cys1-Cys6, Cys2-Cys5 and Cys3-Cys4 disulfide bridges. They are much smaller than other defensins, as they are
constituted of 18 residues.
Among these three subclasses found in mammals, only - and -defensins are found in humans,[62] and although
their disulfide linkages differ, they present similar three-dimensional structures. To be exhaustive, two more subclasses
can be added, corresponding to defensins found in plants and insects:
insect defensins: They contain an -helix domain bound to a -sheet region via a disulfide bridge. Their linkages
differ from those observed in mammals: Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6.[59] They preferentially kill Gram
positive bacteria.[63]
plant defensins: They may contain up to eight cysteins, thus forming up to four disulfide bridges. They are
commonly referred to as thionines. They also contain both -helix and -sheet domains.
Defensins can adopt amphiphilic folds eventually leading to membrane disruption, but while their mechanism of action
among the models proposed has not been clearly identified yet, is thought to be similar to that of the -helical
peptides.[64]

It has to be acknowledged that APs found in humans or in mammals are not restricted to cathelicidins and defensins:
histatins,[20] dermcidins,[65] as well as some anionic peptides[66] are also found in some tissues. Another remark is
that the aforementioned antibacterial peptides do not only kill bacteria, but are also cytotoxic to fungi,[67, 68]
protozoa,[69] malignant cells,[70, 71] and even enveloped viruses like HIV or herpes.[72, 73] Furthermore, a minor
mutation in a given AP can result in a major modification to its activity. Nevertheless, many relationships between
peptide structure and antibacterial activity have been established.

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Table 3 Susceptibilities of three multi-resistant bacteria to selected APs. MIC are expressed in g.ml-1 (adapted from
references [38, 74]).

Peptide E. coli S. aureus P. aeruginosa


Magainin 2 1-7 [75] 8-64 [76] 35-250 [75, 77]
SMAP-29 <1 2.5 [78] 0.8 [78]
LL-37 0.6 [79] 3-10 [79] 2.25 [80]
HNP1-3 0.7 [79] 2.5-25 [76] 10 [81]
hBD1 4-40 [79] > 50 [82] n/a
hBD2 n/a 10 [82] n/a
hBD3 n/a 5 [82] 26.5 [81]

5. Resistance
Bacteria present two types of resistance mechanism towards antibiotics: natural/intrinsic/passive resistance and
learned/acquired/adaptive resistance. Natural resistance of bacteria towards APs does exist and given the ability of
bacteria to adapt to novel conditions, one could think that after extensive use of APs, developing an acquired resistance
would seem very likely. Actually, contrary to antibiotics, cationic peptides do not generally trigger resistance
mechanisms, in spite of a few isolated cases.[83-85] Bacteria do show some mechanisms of resistance towards APs at
different levels:
(i) during the peptide attachment to the membrane,
(ii) during the peptide insertion through the membrane, and
(iii) concerning membrane permeability abilities.

A few bacteria such as Morganella spp. or Serratia spp. for example possess a natural resistance towards APs
because of the constitution of their membrane, which lacks an appropriate density of anionic binding sites. The simplest
way for a bacteria to increase its resistance towards APs is to reduce the negative net charge of its outer membrane.
Gram positive and Gram negative bacteria have developed such mechanisms to highly reduce the electrostatic forces,
thus the AP uptake, inducing the first step of the bacterial killing process.[86] These modifications, identified in many
bacteria, are encoded by a number of genes to modify the teichoic acid of peptidoglycan or the lipid A moiety of LPS in
Gram positive and Gram negative bacteria, respectively. In S. aureus, for instance, the dltA-D gene products transport
positively charged D-alanines from the cytoplasm to the anionic teichoic acids of the bacterial wall.[87] Another weapon
available in the arsenal of bacteria to resist APs is the production of proteases that are able to digest highly positively
charged peptides.[88]Defensins, because of their disulfide bridges, are less sensitive to the action of proteases. The
modification of membrane fluidity also accounts for an enhanced resistance. Gram negative bacteria increase the
hydrophobic interactions within their outer membrane, consequently the membrane fluidity is decreased and so is its
affinity towards APs.[89] AP insertion and pore formation are hindered by this increased hydrophobicity of the
membrane. It has also been shown that ATP-Binding Casette (ABC) transporters import APs into the bacteria, while
efflux pumps export them. Overexpression of these efflux pumps can explain resistance to APs.[90] Formation of
biofilms[91] and synthesis of molecules to bind APs in order to neutralize[92] them are also mechanisms that have been
reported.
Moreover, to be competitive, bacteria produce antimicrobial peptides too, called bacteriocins, which are generally
more potent than conventional APs. To prevent its membrane to be disrupted by its own peptides, the bacteria has
developed protective mechanisms by carrying resistance genes expressed as efflux pumps, specifically sequestering
enzymes or competitors for target binding.[93]
Nevertheless, many studies reached the conclusion that it would be difficult for bacteria to develop resistance against
APs.[94-96] Indeed, for instance, it takes 30 passages of P. aeruginosa in sub-MIC peptide concentration to increase its
resistance by four-fold at most[97] whereas resistance to gentamicin, an aminoglycoside antibiotic, increases by 190-
fold.[94] On the other hand, another study showed that after 700 passages of an AP derived from magainin with a
slowly increasing concentration, the MIC increased from 1 g.ml-1 to 500 g.ml-1.[98] In conclusion, one has to bear in
mind that the primary target of an AP is the bacterial membrane. To grow a resistance against it, the bacteria would
have to redesign its whole membrane, a very lengthy process. This argument, added to the fact that APs are part of the
innate immune system, meaning that bacteria have had a huge time-span to adapt to their killing mechanisms, makes it
very unlikely for a resistance phenomenon to suddenly arise.
However, it has also to be acknowledged that among microbes, only bacteria show resistance mechanisms against
APs.

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6. Use of Antibacterial Peptides as therapeutic agents


We have seen that APs have broad spectra of activity, are efficient towards multi-resistant bacteria and are not hindered
by resistance, which might make them potential replacement for conventional antibiotics. Compared to the latter, APs
kill bacteria rapidly, can involve multiple targets,[13] and their minimal inhibitory concentrations often coincide with
their minimal bactericidal concentrations (with less than a two-fold difference). However, their potency against the most
susceptible bacteria is not as strong and peptides are generally considered to be poor drug candidates[99] because of
their low oral bioavailability and their propensity to be rapidly metabolized. Moreover, their synthesis is often
challenging with high associated R&D costs.
As pointed out above, today over a thousand APs have been discovered, either by being isolated from tissues or
identified by sequence analyses. This diversity of structures, mechanisms of action and spectra of activity represents a
gold mine for the design of THE perfect drug. To this purpose, many research teams from academia and industry
contribute to the understanding of structure-activity relationships, to the development of potent synthetic mimics,
The design of new APs is an infinite task when considering that an AP can be constituted of 3 to more than 50 amino
acid residues. Indeed the number of possible combinations for a given AP of N residues is 20N. Thus, the starting point
to develop an efficient AP is to identify a naturally potent one and operate some modifications to optimize it. The most
important requirements to develop/enhance an AP are the following: selective toxicity for the bacterial target, broad
spectrum of activity, formulation with high chemical stability, few side effects, minimal risk of bacterial resistance,
favorable costs.
Broad spectrum of activity:
Although APs exhibit significant and broad in vitro activity, it often significantly decreases under physiological salt and
serum conditions in vivo. Besides, it has been described that APs show the same activity towards wild-type and resistant
strains of bacteria.[100] Natural sources produce APs in low quantities/concentration with a limited spectrum of activity
compared to enhanced synthetic APs.
Toxicity:
APs can be toxic to eukaryotic cells, as their mechanism of action principally relies on interaction with membranes.
This potential toxicity also represents a key obstacle for their clinical application. However, human cells are globally
resistant to APs at reasonable concentrations, but it should be mentioned that venom from bees, wasps and scorpions are
actually composed of APs, toxic to humans. At higher concentration, some defensins are cytotoxic to mammalian
cells.[101-103]
Costs:
This is the principal obstacle to overcome. Producing one gram of an AP can cost up to 400US$, whereas for a
conventional antibiotic, this price can be under 1US$.[104] Commercial-scale production platforms to synthesize APs
are urgently needed.

The use of cationic antibacterial peptides as therapeutic agents has been established and validated.[105] Several
companies are currently attempting to commercialize AP-based drugs, but as of today, none have reached the market.
Nevertheless, many anti-microbial peptides are currently being tested in clinical trials.
One of the first generation developed AP is Pexiganan (or MSI-78). This peptide designed in the 90s derives from
magainin-2 and is composed of 22 amino acids. Although Pexiganan entered Phase III trials, proved to be effective in
wound healings and did not show any notable toxicity or side-effects,[106] it was rejected by the Food and Drug
Administration (FDA) in 1999. Indeed its efficiency towards infected diabetic foot ulcers did not offer any
improvement over the conventional treatment with ofloxacin, a fluoroquinolone antibiotic.[107] And yet, Pexiganan
showed an in vitro activity against Gram positive and Gram negative bacteria much more efficient than ofloxacin.[108,
109] Iseganan (IB367, a pig protegrin synthetic analog)[110] and Neuprex[111] encountered the same fate as
Pexiganan, as they did not pass the Phase III trials.
MX-226 and MX-594NA, bovine indolicidin-based APs variants from Migenix (Canada), have recently shown
efficiency in Phase III clinical studies. MX-226 was developed for the prevention of catheter related infections, whereas
MX-594NA was developed for the treatment of acnea vulgaris. Xoma (USA) has XOMA629 under preclinical studies,
an AP deriving from the human BPI protein, which showed promising activities against skin bacteria. P113, developed
by Demegen (USA), derives from histatin and features 12 amino acid residues. This AP showed excellent in vitro
activities against Gram positive and Gram negative bacteria and is to be used as a mouth rinse product, to fight e.g.
gingivitis.[112]
Plectasin, a fungal defensin, is currently used and developed by Novozymes.[113] This AP is currently under
preclinical development and was shown to be active against Streptococcus pneumoniae. Plectasin might be the first
member of a new class of AP based antibiotics.

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7. Concluding remarks
In the close vicinity of a membrane surface, structure-activity relationships have clearly been established for APs in vivo, but
much remains to be learned in order to ideally and successfully replace conventional antibiotics by peptides. The potential to
become an important class of drugs to add to the arsenal against bacteria fight does exist for APs, but many hurdles still have
to be overcome. Aside from the production cost problems, further work to understand the precise mechanism of action of any
AP of interest, to circumvent possible resistance mechanisms and possible toxicity is of primary importance. In the worst
case scenario, APs could work in synergy with antibiotics to lower bacterial resistance barriers. Besides, their
therapeutic use is not limited to bacteria, since APs have shown to fight viruses and fungi as well.

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